月份：2022年4月

Dojindo,Bacstain-CTC快速染色试剂盒-用于流式细胞术/100/BS01

Product Description

使用琼脂板形成菌落是一种非常常见且可靠的细菌细胞计数方法。然而，形成一个殖民地需要相当长的时间。因此，已经开发了替代的检测方法。细菌特异性基因扩增方法，如 PCR、LAMP 和细胞核染色非常快速，但这些方法也可以计算死细菌。因此，检测活细胞功能对于确定样本中活细菌的实际数量至关重要。四唑鎓盐可用于检测细菌细胞或线粒体的呼吸活动。 CTC 是一种四唑盐，通过这种呼吸活动被还原，在细胞表面形成荧光 CTC 甲臜。因此，CTC 用于需氧活菌的特异性染色，可应用于难以培养的细菌（VNC：活但不可培养）。 CTC 通过电子转移系统形成荧光甲臜。然而，单独的 CTC 不足以对单个细胞进行染色。因此，CTC-Rapid Staining Kit 含有一种增强试剂，可提高 CTC 染色效率。与仅使用 CTC 染色相比，该染色试剂盒能够快速、灵敏地对微生物进行染色。 CTC 甲臜染料的最大激发波长为 430 nm 或 480 nm，发射波长为 630 nm。

General Staining Protocol

显微镜检测1。 离心细菌培养物并去除上清液，然后用PBS(-)重悬细菌沉淀。2． 添加 CTC + 增强试剂-B。 在 37°C 下孵育 1 小时。 3. 准备一张幻灯片并通过 B 激发滤光片组检测荧光。

流式细胞仪检测1。 离心细菌培养物并去除上清液，然后用PBS(-)重悬细菌沉淀。2． 添加 CTC + 增强试剂-A。 在 37°C 下孵育 1 小时。 3. 用流式细胞仪分析细胞：488 nm 激发，630 nm 发射。

1. A. Hiraishi, et al., An Improved Redox Dye-Staining Method Using 5-Cyano-2,3-Ditoryl Tetrazolium Chloride for Detection of Metabolically Active Bacteria in Activated Sludge. Microbes Environ. 2004;19:61-70.2. A. Kitaguchi, et al., Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization Following Formazan Reduction. Appl Environ Microbiol. 2005;71:2748-2752.

FACS DataE. coli

B. Subtilis

Candida albicans

E. faecalisFig. 2 CTC staining of various microorganisms with and without Enhancing reagent.

Experimental ExampleObservation by Microscopy

Fig. 3 E. coli staining (left) and L. casei staining (right) with CTC and DAPI. Bacterial cells were stained with CTC Rapid Staining Kit first, and then 1 μl of DAPI solution was added. The cells were incubated at room temperature for 5 min. Formaldehyde fixation with 1-4% formaldehyde can be performed before DAPI staining.

Dojindo,16-羟基-1-十六烷基硫醇/10/H394

Product Description

羟基烷硫醇用作金表面上的稀释试剂以控制反应基团的密度，或用作封闭剂以防止分析物在表面上的非特异性结合。新开发的 16-Hydroxy-1-hexadecanethiol 具有 16 个碳链，是市场上羟基烷硫醇中最长的烷硫醇。总共有 6 种不同的羟基烷硫醇，包括可用于金表面改性的羟基-EG6-十一烷硫醇和羟基-EG3-十一烷硫醇。当应用 16-Amino-1-hexadecanethiol 或 15-Carboxy-1-pentadecanethiol 时，16-Hydroxy-1-hexadecanethiol 用于制备均匀且高度定向的 SAM。 Herne 和他的同事在金表面上制造了硫醇衍生的单链 DNA (HS-ss-DNA) 和 6-Hydroxy-1-己硫醇的混合 SAM，以防止 HS-ss-DNA 的非特异性吸附。 Perez-Luna 和其他人在金表面上制作了生物素末端硫醇和 11-羟基-1-十一烷硫醇的混合 SAM。它们阻止了野生型链霉亲和素和链霉亲和素突变体的非特异性吸附。 Dubrovsky 及其同事使用 11-Hydroxy-1-十一硫醇控制了蛋白质在镀金硅胶表面上的非特异性吸附。他们提到了镀金硅胶在制备用于生物测定的明确的、表面功能化的支持物方面的有用性。

Dojindo,R-藻红蛋白标记试剂盒-NH2/3/LK23,藻胆蛋白是源自蓝细菌和真核藻类的荧光蛋白

Product Description
藻胆蛋白是源自蓝细菌和真核藻类的荧光蛋白。 它们的荧光远高于荧光素和罗丹明等化学荧光探针。 R-藻红蛋白（R-PE）是一种藻胆蛋白，在578 nm左右发出红色荧光，在488 nm处可被激发（图1）。 由于这种高荧光，藻胆蛋白标记的抗体和其他分子可以在流式细胞术和免疫染色中提供更高的灵敏度。 R-藻红蛋白标记试剂盒-NH2 用于简单快速地制备 R-PE 标记的 IgG（图 2）。 NH2-reactive R-PE（本试剂盒的一个组成部分）具有活化的酯基，无需任何活化过程即可轻松与目标分子的氨基形成共价键。 该套件中的过滤管允许快速更换缓冲液并浓缩样品 IgG 溶液。 该试剂盒包含 R-PE 标记所需的所有试剂，包括偶联物的储存缓冲液。
 

Precaution♦ 使用本试剂盒标记的蛋白质的分子量应大于 50,000。♦ 标记过程中，IgG 或 R-藻红蛋白结合的 IgG 始终在过滤管的膜上。♦ 如果 IgG 溶液中含有其他具有 分子量大于 10,000，例如 BSA 或明胶，在用此试剂盒标记 R-藻红蛋白之前纯化 IgG 溶液。 IgG 溶液可通过 IgG 纯化试剂盒（本试剂盒未提供）进行纯化。♦ 如果 IgG 溶液含有少量不溶性物质，则将溶液离心并使用上清液进行标记。

1. K.R. McCarthy, A. Watanabe, M. Kuraoka, K.T. Do, C.E. McGee, G.D. Sempowski, T.B. Kepler, A.G. Schmidt, G. Kelsoe and S.C. Harrison, “Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires”, Immunity., 2018, 48, (1), 174.2. K.Y Su, A. Watanabe, C.H. Yeh, G. Kelsoe, M. Kuraoka, “Efficient Culture of Human Naive and Memory B Cells for Use as APCs”, J. Immunol.., 2016, 197, (10), 4163.3. T. Tsujikawa, T. Yaguchi, G. Ohmura, S. Ohta, A. Kobayashi, N. Kawamura, T. Fujita, H. Nakano, T. Shimada, T. Takahashi, R. Nakao, A. Yanagisawa, Y. Hisa, Y. Kawakami, “Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma”, Int. J. Cancer., 2013, 132, (12), 2755.4. Y. Inaguma, Y. Akahori, Y. Murayama, K. Shiraishi, S. Tsuzuki-Iba, A. Endoh, J. Tsujikawa, A. Demachi-Okamura, K. Hiramatsu, H. Saji, Y. Yamamoto, N. Yamamoto, Y. Nishimura, T. Takahashi, K. Kuzushima, N. Emi, Y. Akatsuka, “Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H”, Gene Ther.., 2014, 21, (6), 575.

Can I use this kit for other proteins?

Yes, if the molecular weight is higher than 50,000, and it has a reactive primary or secondary amino group. Follow the protocol for IgG labeling with 0.5-1 nmol of sample protein.

How many R-PE molecules per IgG are introduced?

The average number of R-PE molecule per IgG is 1 to 2.

Does unconjugated NH2-reactive R-PE still have an activated ester after the labeling reaction to IgG?

No. It is completely hydrolyzed during the reaction.

Does NH2-reactive R-PE form an oligomer during the labeling reaction?

No. Since all amino groups of NH2-reactive R-PE are blocked, no oligomerization is possible.

What is the minimum amount of IgG that can be labeled with LK23-10?

The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG.

Do I have to use WS buffer included with the kit?

Yes. Use the WS buffer to prepare a stock solution of the conjugate. However, you can choose any kind of buffer appropriate to dilute the conjugate stock solution for your experiment.

How long is the conjugate stable?

If you store at 4ºC, it is stable for over 2 months. For longer storage, add 100% volume of glycerol, aliquot, and store at -20ºC. However, please note that the stability depends on the protenitself.

Dojindo,碱性磷酸酶标记试剂盒-NH2/3/LK12,NH2 反应性 ALP 是该试剂盒的组成部分

碱性磷酸酶标记试剂盒-NH2主要用于制备碱性磷酸酶标记的IgG用于酶免疫分析（EIA）和制备碱性磷酸酶标记的抗原用于竞争性EIA。 NH2 反应性 ALP 是该试剂盒的组成部分，可与蛋白质或其他分子的氨基反应（图 1-15）。 该试剂盒包含标记过程所需的所有试剂，包括存储缓冲液。 NH2 反应性 ALP 无需任何激活过程即可与靶分子形成共价键。 当碱性磷酸酶标记的 IgG 用于 EIA 时，NH2 反应性 ALP 的标记效率高到足以消除标记后的任何纯化过程。 如果标记后需要高纯度偶联物，只需使用亲和柱或凝胶渗透柱即可。 标记小分子时，可以使用该套件中包含的过滤管去除多余的分子。

Fig. 1 IgG labeling reaction of NH2-reactive alkaline phosphatase

Precaution♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.♦ The molecular weight of the small amine compound to be labeled with this kit should be smaller than 5,000.♦ IgG or alkaline phosphatase-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with molecular weight larger than 10,000, such as BSA or gelatin, purify the IgG solution prior to labeling alkaline phosphatase with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

1. B. Pandey, A.V. Demchenko, K.J. Stine, “Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen”, Microchim Acta., 2012, 179, (1-2), 71.2. M. Watanabe, I. Takemasa, N. Kaneko, Y. Yokoyama, E. Matsuo, S. Iwasa, M. Mori, N. Matsuura, M. Monden, and O. Nishimura, “Clinical significance of circulating galectins as colorectal cancer markers”, Oncol. Rep.., 2011, 25, (5), 1217.3. Y. Matsumae, Y. Takahashi, H. Shiku and T. Matsue, “Quantitative Real‐Time Monitoring of Antibody‐Induced Internalization of Epidermal Growth Factor Receptor on Single Living Mammalian Cells Using Scanning Electrochemical Microscopy”, ChemElectroChem., 2018, 5, (20), 3096.

Western blotting

Fig. 2 Western blotting of Alkaline phosphatase-conjugated antibody prepared by Alkaline phosphatase Labeling Kit-NH2.1: antigen 50 ng2: antigen 10 ng3: antigen 2 ng

A target protein (antigen) was detected with ALP-labeled antibody prepared by Alkaline Phosphatase Labeling Kit-NH2 after it was run with SDS-PAGE and transferred to a nitrocellulose membrane. A target protein was detected with a chemiluminescence substrate for alkaline phosphatase after the treatment with 25,000 times dilution of ALP-labeled primary antibody.

我可以将此套件用于 Fab 或 Fab Elabeling 吗？
是的，您可以使用此套件标记 Fab 和 Fab E。偶联物的回收率应在 80% 以上。
我可以将此试剂盒用于其他蛋白质或肽吗？
是的，如果分子量高于 50,000 或低于 5,000，并且它具有反应性伯氨基或仲氨基。如果分子量高于 50,000，按照 IgG 的标记方案进行，LK12-10 使用 0.5-1 nmol 的样品蛋白。如果分子量低于 5,000，请遵循小分子的标记协议。如果分子量低于 50,000 但高于 5,000，请联系我们的客服 info@dojindo.com 或 1-877-987-2667 了解更多信息。
我可以使用该试剂盒标记寡核苷酸或寡肽吗？
是的，如果分子量小于 5,000，并且它具有反应性伯氨基或仲氨基。遵循小分子的标记协议。
可以用 LK12-10 标记的 IgG 的最低量是多少？
最小量为 50 μg。 50 μg 和 200 μg IgG 的灵敏度和背景没有显着差异。尽管使用该试剂盒仍可标记 10 μg IgG，但背景会更高。
每个 IgG 引入多少碱性磷酸酶分子？
每个 IgG 的平均碱性磷酸酶分子数为 1 至 3。
与 IgG 标记反应后，未结合的 NH2 反应性碱性磷酸酶是否仍有活化酯？
不会。NHS 在反应过程中完全水解。
NH2 反应性碱性磷酸酶在标记反应过程中会形成寡聚体吗？
不会。由于 NH2 反应性碱性磷酸酶的所有反应性氨基都被封闭，因此不可能发生低聚反应。
我必须使用套件中包含的存储缓冲液吗？
不，您不必使用套件中的存储缓冲液。您可以选择适合您实验的任何类型的缓冲液。
储存缓冲液是否包含动物产品或聚合物？
不，储存缓冲液不含任何动物产品、聚合物或重金属离子。

Dojindo,Cellstain-DAPI溶液/1/D523,DAPI 是一种 AT 序列特异性 DNA 嵌入剂

DAPI 是一种 AT 序列特异性 DNA 嵌入剂，它像 Hoechst 染料一样在双螺旋的小沟处附着在 DNA 上。 尽管 DAPI 不能透过活细胞膜，但它会通过受干扰的细胞膜对细胞核进行染色。 DAPI 具有较高的光漂白耐受性水平。 DAPI 用于检测酵母中的线粒体 DNA、叶绿体 DNA、病毒 DNA、支原体 DNA 和染色体 DNA。 DAPI-DNA 复合物的激发和发射波长分别为 360 nm 和 460 nm。

Staining Procedure1.Prepare 10-50 μM DAPI solution with PBS or an appropriate buffer.a)2.Add DAPI solution with 1/10 of the volume of cell culture medium to the cell culture.b)3.Incubate the cell at 37oC for 10-20 min.4.Wash cells twice with PBS or an appropriate buffer.5.Observe the cells using a fluorescence microscope with 360 nm excitation and 460 nm emission filters.

a) Since DAPI may be carcinogenic, extreme care is necessary during handling.b) Or you may replace the culture medium with 1/10 concentration of DAPI buffer solution.

1. W. Schnedl, et al., DIPI and DAPI: Fluorescence Banding with Only Negligible Fading. Hum Genet. 1977;36:167-172.2. I. W. Taylor, et al., An Evaluation of DNA Fluochromes, Staining Techniques, and Analysis for Flow Cytometry. I. Unperturebed Cellpopulations. J Histochem Cytochem. 1980;28:1224-1232.3. F. Otto, et al., A Comparative Study of DAPI, DIPI, and Hoechst 33258 and 33342 as Chromosomal DNA Stains. Stain Technol. 1985;60:7-11.4. N. Poulin, et al., Quantitative Precision of an Automated Image Cytometric System for the Measurement of DNA Content and Distribution in Cells Labeled with Fluorescent Nucleic Acid Stains. Cytometry. 1994;16:227-235.5. M. Kawai, et al., Rapid Enumeration of Physiologically Active Bacteria in Purified Water Used in the Pharmaceutical Manufacuturing Process. J Appl Microbiol. 1999;86:496-504.

Dojindo,ODPA/100/O407,膦酸衍生物同样在金属氧化物上形成SAM

膦酸衍生物用于氧化金属的表面改性，如 Al2O31)、TiO22)、ZrO23)、SiO24)、Mica5)、不锈钢(SS316L)6)、镍钛诺7)、羟基磷灰石8)、AgO9)、ZnO10)、ITO11、12 ). 长期以来，有机硅烷一直被用于在金属氧化物上形成自组装单层 (SAM)。 然而，由于试剂之间的稳定性和聚合性差，在应用中并不总是适用。 另一方面，膦酸衍生物同样在金属氧化物上形成SAM，尽管它们是非常稳定的化合物。 此外，据报道，膦酸衍生物使用形成比有机硅烷更稳定和致密的 SAM。 克劳克等。 人。 和 Sekitani 等。 人。 显示 Al2O3 上的烷基膦酸酯 SAM 比三氯硅烷衍生物 SAM 作为有机晶体管的导体膜更有用13）。

1) T. Hauffman, O. Blajiev, J. Snauwaert, C. van Haesendonck, A. Hubin, H. Terryn,  EStudy of the self-assembling of n-octylphosphonic acid layers on aluminum oxide E Langmuir, 2008, 24 (23), 13450.2) B. M. Silverman, K. A. Wieghaus, J. Schwartz, “Comparative properties of siloxane vs phosphonate monolayers on a key titanium alloy E Langmuir, 2005, 21(1), 225.3) W. Gao, L. Reven, “Solid-state NMR-studies of self-assembled monolayers E Langmuir 1995, 11 (6), 1860.4) E. L. Hanson, J. Schwartz, B. Nickel, N. Koch, M. F. Danisman, “Bonding self-assembled, compact organophosphonate monolayers to the native oxide surface of silicon E J. Am. Chem. Soc. 2003, 125 (51), 16074.5)J. T. Woodward, A. Ulman, D. K. Schwartz, “Self-assembled monolayer growth of octadecylphosphonic acid on mica E Langmuir 1996, 12 (15), 3626.6)A. Raman, M. Dubey, I. Gouzman and E. S. Gawalt, “Formation of self-assembled monolayers of alkylphosphonic acid on the netive oxide surface of SS316L E Langmuir, 2006, 22, 6469.7) R. Quinones and E. S. Gawalt, “Polystyrene formation on monolayer-modified nitinol effectively controls corrosion E Langmuir, 2008, 24, 10858.8) S. C. D’Andrea and Al. Y. Fadeev, “Covalent surface modification of calcium hydroxyapatite using n-alkyl- and n-fluoroalkylphosphonic acids EB>, Langmuir, 2003, 19, 7904.9) Y. T. Tao, C. Y. Huang, D. R. Chiou, L. J. Chens, “Infrared and atomic force microscopy imaging study of the reorganization of self-assembled monolayers of carboxylic acids on silver surface E Langmuir, 2002, 18, 8400.10) B. Zhang, T. Kong, W. Xu, R. Su, Y. Gao and G. Cheng, “Surface functionalization of zinc oxide by carboxyalkylphosphonic acid self-assembled monolayers E Langmuir, 2010, 26(6), 4514.11) A. Sharma, B. Kippelen, P. J. Hotchkiss and S. R. Marder, “Stabilization of the work function of indium tin oxide using organic surface modifiers in organic light-emitting diodes E Appl. Phys. Lett., 2008, 93, 163308.12) A. Pulsipher, N. P. Westcott, W. Luo, and M. N. Yousaf, “Rapid in situ generation of two patterned chemoselective surface chemistries from a single hydroxy-terminated surface using controlled microfluidic oxidation E J. Am. Chem. Soc., 2009, 131(22), 762613) a) H. Klauk, U. Zschieschang, J. Pflaum, M. Halik,  E/SPAN>Ultralow-power organic complementary circuits E Nature, 2007, 445, 745. b) T. Sekitani, Y. Noguchi, U. Zschieschang, H. Klauk, T. Someya, “Organic transistors manufactured using inkjet technology with subfemtoliter accuracy E Proc. Natl. Acad. Sci. USA, 2008, 105, 4976

Dojindo,Cellstain-AO溶液/1/A430,AO 与单链 DNA,RNA 形成复合物以发出红色荧光

吖啶橙 (AO) 与双链 DNA 形成复合物，发出绿色荧光（图 1）。 AO 还与单链 DNA 或 RNA 形成复合物以发出红色荧光。 一分子AO嵌入三对双链DNA碱基，发出绿色荧光，最大波长为526 nm（激发波长502 nm）。 一个 AO 分子还可以与单链 DNA 或 RNA 的一个磷酸基团相互作用，形成一个聚集或堆叠的结构，该结构发出最大波长为 650 nm（激发 460 nm）的红色荧光。 因此，AO 可用于检测双链 DNA 和单链 DNA 或 RNA。 它可以通过氩激光激发或流式细胞术同时测定 DNA 和 RNA。

Staining Procedure1.Prepare 10-50 μM AO solution with PBS or an appropriate buffer.a)2.Add AO solution with 1/10 of the volume of cell culture medium to the cell culture.b)3.Incubate the cell at 37ºC for 10-20 min.4.Wash cells twice with PBS or an appropriate buffer.5.Observe the cells under a fluorescence microscope with 500 nm excitation and 530 nm emission filters.

a) Since AO may be carcinogenic, extreme care is necessary during handling.b) You may replace the culture medium with 1/10 concentration of AO buffer solution.

1. I. W. Taylor, et al., An Evaluation of DNA Fluochromes, Staining Techniques, and Analysis for Flow Cytometry. I. Unperturebed Cellpopulations. J Histochem Cytochem. 1980;28:1224-1232.2. N. Miyoshi, et al., Fluorescence Lifetime of Acridine Orange in Sodiu Dodecyl Sulfate Premicellar Solutions. Photochem Photobiol. 1988;47:685-688.3. A. K. El-Naggar, et al., Single- and Double-stranded RNA Measurements by Flow Cytometry in Solid Neoplasms. Cytometry. 1991;12:330-335. 4. Y. Miyakoshi, et al., The Frequencies of Micronuclei Induced by Cisplatin in Newborn Rat Astrocytes Are Increased by 50-Hz, 7.5- and 10-mT Electromagnetic Fields. Environ Health and Prev Med. 2005;10:138-143.

Dojindo,Cellstain- Double Staining Kit/1/CS01,Cellstain-双重染色试剂盒

Cellstain-Double Staining Kit 用于同时对活细胞和死细胞进行荧光染色。该试剂盒包含 Calcein-AM 和碘化丙啶 (PI) 溶液，分别用于染色活细胞和死细胞（图 1）。 Calcein-AM 是一种钙黄绿素的乙酰氧基甲酯，具有高度亲脂性和细胞膜渗透性。虽然 Calcein-AM 本身不是荧光分子，但 Calcein-AM 在活细胞中通过酯酶产生的 calcein 会发出强烈的绿色荧光（激发：490 nm，发射：515 nm）。因此，Calcein-AM 仅对活细胞染色。另一方面，细胞核染色染料 PI 不能通过活细胞膜。它通过死细胞膜的无序区域到达细胞核，并与细胞的DNA双螺旋插入并发出红色荧光（激发：535 nm，发射：617 nm）。由于 calcein 和 PI-DNA 都可以在 490 nm 下激发，因此可以使用荧光显微镜同时监测活细胞和死细胞。在 545 nm 激发下，只能观察到死细胞（图 2）。由于最佳染色条件因细胞系而异，我们建议单独确定合适的 PI 和 Calcein-AM 浓度。请注意，PI被怀疑具有高度致癌性；需要小心处理。

Optimization ProcedureThe following steps may be necessary to determine the suitable concentration of each reagent:

1. Prepare dead cells by 10 minutes incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30 minutes incubation in 70% ethanol.2. Stain dead cells with 0.1-10 μM PI solution to find a PI concentration that stains the nucleus only, not the cytosol.3. Stain dead cells with 0.1-10 μM Calcein-AM solution to find a Calcein-AM concentration that does not stain the cytosol. Then stain viable cells with that Calcein-AM solution to check whether the viable cell can be stained.

1. L. S. D. Clerck, et al., Use of Fluorescent Dyes in the Determination of Adherence of Human Leucocytes to Endothelial Cells and the Effects of Fluorochromes on Cellular Function. J Immunol Methods. 1994;172:115-124.2. E. S. Kaneshiro, et al., Reliability of Calcein Acetoxy Methyl Ester and Ethidium Homodimer or Propidium Iodide for Viability Assessment of Microbes. J Microbiol Methods. 1993;17:1-16.3. N. G. Papadopoulos, et al., An Improved Fluorescence Assay for the Determination of Lymphocyte-Mediated Cytotoxicity Using Flow Cytometry. J Immunol Methods. 1994;177:101-111.4. M. Adler, et al., Cytotoxic actions of the heavy metal chelator TPEN on NG108-15 neuroblastoma-glioma cells. Neurotoxicology. 1999;20:571-582.5. P. G. Bush, et al., Viability and volume of in situ bovine articular chondrocytes-changes following a single impact and effects of medium osmolarity. Osteoarthritis Cartilage. 2005;13:54-65.

Dojindo,M-EG3-UPA/100/M457,M-EG-UPA含有低聚乙二醇基团末端的烷基膦酸衍生物

膦酸衍生物用于氧化金属的表面改性，如 Al2O31)、TiO22)、ZrO23)、SiO24)、Mica5)、不锈钢(SS316L)6)、镍钛诺7)、羟基磷灰石8)、AgO9)、ZnO10)、ITO11、12 ). 长期以来，有机硅烷一直被用于在金属氧化物上形成自组装单层 (SAM)。 然而，由于试剂之间的稳定性和聚合性差，在应用中并不总是适用。 另一方面，膦酸衍生物同样在金属氧化物上形成SAM，尽管它们是非常稳定的化合物。 此外，据报道，膦酸衍生物使用形成比有机硅烷更稳定和致密的 SAM。 克劳克等。 人。 和 Sekitani 等。 人。 显示 Al2O3 上的烷基膦酸 SAM 比三氯硅烷衍生物 SAM 更适合作为有机晶体管的导体膜 13)。M-EG-UPA 是一种含有低聚乙二醇基团末端的烷基膦酸衍生物。

1. T. Hauffman, O. Blajiev, J. Snauwaert, C. van Haesendonck, A. Hubin, H. Terryn,  EStudy of the self-assembling of n-octylphosphonic acid layers on aluminum oxide E Langmuir, 2008, 24 (23), 13450.2. B. M. Silverman, K. A. Wieghaus, J. Schwartz, “Comparative properties of siloxane vs phosphonate monolayers on a key titanium alloy E Langmuir, 2005, 21(1), 225.3. W. Gao, L. Reven, “Solid-state NMR-studies of self-assembled monolayers E Langmuir 1995, 11 (6), 1860.4. E. L. Hanson, J. Schwartz, B. Nickel, N. Koch, M. F. Danisman, “Bonding self-assembled, compact organophosphonate monolayers to the native oxide surface of silicon E J. Am. Chem. Soc. 2003, 125 (51), 16074.5. J. T. Woodward, A. Ulman, D. K. Schwartz, “Self-assembled monolayer growth of octadecylphosphonic acid on mica E Langmuir 1996, 12 (15), 3626.6. A. Raman, M. Dubey, I. Gouzman and E. S. Gawalt, “Formation of self-assembled monolayers of alkylphosphonic acid on the netive oxide surface of SS316L E Langmuir, 2006, 22, 6469.7. R. Quinones and E. S. Gawalt, “Polystyrene formation on monolayer-modified nitinol effectively controls corrosion E Langmuir, 2008, 24, 10858.8. S. C. D’Andrea and Al. Y. Fadeev, “Covalent surface modification of calcium hydroxyapatite using n-alkyl- and n-fluoroalkylphosphonic acids Estrong>, Langmuir, 2003, 19, 7904.9. Y. T. Tao, C. Y. Huang, D. R. Chiou, L. J. Chens, “Infrared and atomic force microscopy imaging study of the reorganization of self-assembled monolayers of carboxylic acids on silver surface E Langmuir, 2002, 18, 8400.10. B. Zhang, T. Kong, W. Xu, R. Su, Y. Gao and G. Cheng, “Surface functionalization of zinc oxide by carboxyalkylphosphonic acid self-assembled monolayers E Langmuir, 2010, 26(6), 4514.11. A. Sharma, B. Kippelen, P. J. Hotchkiss and S. R. Marder, “Stabilization of the work function of indium tin oxide using organic surface modifiers in organic light-emitting diodes E Appl. Phys. Lett., 2008, 93, 163308.12. A. Pulsipher, N. P. Westcott, W. Luo, and M. N. Yousaf, “Rapid in situ generation of two patterned chemoselective surface chemistries from a single hydroxy-terminated surface using controlled microfluidic oxidation E J. Am. Chem. Soc., 2009, 131(22), 762613. a) H. Klauk, U. Zschieschang, J. Pflaum, M. Halik,  E/span>Ultralow-power organic complementary circuits E Nature,2007, 445, 745. b) T. Sekitani, Y. Noguchi, U. Zschieschang, H. Klauk, T. Someya, “Organic transistors manufactured using inkjet technology with subfemtoliter accuracy E Proc. Natl. Acad. Sci. USA, 2008, 105, 4976

Dojindo,Cellular Senescence Plate Assay Kit-SPiDER-ßGal/100/SG05,细胞衰老板检测试剂盒

本产品是一种简单的板式检测试剂盒，用于检测衰老相关的β-半乳糖苷酶（SA-β-gal）活性，用作衰老细胞的标志物。 该试剂盒只需将检测 β-半乳糖苷酶的试剂 SPiDER-βGal 加入 96 孔板中，即可量化 SA-β-gal 活性，并可以评估多个样本。 当通过细胞计数、核酸定量（相关产品）或蛋白质定量获得的结果进行标准化时，使用该试剂盒获得的测量值可用于根据细胞数评估 SA-β-gal 活性。

Simply Quantify Senescent Cells:Cells prepared in advance are lysed in the buffer supplied with this kit.Fluorescence intensity is obtainable according to SA-β-gal activity simply by adding the fluorescent substrate SPiDER-βGal to the cell lysate. Even when you prepare cells in 100 mm dishes or others, fluorescence intensity can be measured by transferring cell lysate in 96 well plates after cell lysis.

 

Correlation with imaging data:Imaging assessments of WI-38 cells at different passage levels were performed with this Plate Assay Kit and the Cellular Senescence Detection Kit – SPiDER-βGal

As a result, it was confirmed that in both kits, SA-β-gal staining increased in the high-passage WI-38 cells.Bear in mind that although initial cell seeding densities are the same, cell densities at the time of plate assay differ due to low proliferation rate of senescent cells at higher passage levels. Therefore, in this experiment, we used SA-β-Gal activity values normalized by the results obtained using the Cell Count Normalization Kit (coming soon) in which cell number is determined by a nuclear marker.

Plate Assay<Condition>Ex. 535nm / Em. 580nm

Imaging data<Condition>Green: Ex. 488nm / Em. 500-600nm (SA-β-Gal staining with Cellular Senescence Detection Kit – SPiDER-βGal(Item# SG04))Blue: Ex. 405nm / Em. 450-495nm (Nuclear staining with -Cellstain- DAPI solution(Item# D523))

Evaluation doxorubicin-treated with cells:We performed plate assays using this kit with WI-38 cells after adding doxorubicin which increases the production of mitochondrial reactive oxygen species (ROS). As a result, it was confirmed that the addition of doxorubicin to WI-38 cells caused increased staining intensity of SA-β-gal due to DNA damage-induced senescence.

Ex. 535nm / Em. 580nm

Precautions when using this kit:Cell counts may need to be normalized. When cells are analyzed in a microplate, the results obtained may sometimes differ depending on cell numbers per well.In such cases, normalization of the measured values obtained from cell counting and total protein will be necessary. In this kit, cell numbers can be easily measured by the fluorescence intensity induced by a reagent added to cell culture medium for staining nuclei.

Dojindo,Biotin Sulfo-OSu/10/B319,Biotin-(AC5)2-OSu用作 Biotin Labeling Kit-NH2 中的生物素标记剂

抗生物素蛋白-生物素系统在免疫学和组织化学中有许多应用。抗生物素蛋白和生物素之间的相互作用非常强，解离常数约为 10-15 M。生物素通常添加到一抗或二抗中，例如抗 IgG 和抗 IgM。在用生物素标记的抗体制备抗原-抗体复合物后，使用酶或荧光素标记的抗生物素蛋白或链霉抗生物素蛋白对抗原进行比色或荧光检测。琥珀酰亚胺酯生物素在 pH 7-9 下与伯胺和仲胺（例如氨基酸和蛋白质）反应。琥珀酰亚胺酯与游离胺基反应生成稳定的酰胺键。琥珀酰亚胺基生物素试剂必须溶解在 DMSO、DMF 或酒精中。用 DMSO 制备的储备溶液在 -20ºC 下可稳定数月。磺基琥珀酰亚胺生物素试剂可溶于水，因此无需使用 DMF 或 DMSO 等有机溶剂。使用具有较长间隔物的生物素（如 Biotin-(AC5)2-OSu 或 Biotin-(AC5)2-Sulfo-OSu）制备的 IgG 具有更好的信噪比。较长的间隔使链霉亲和素或抗生物素 IgG 能够在没有结构抑制的情况下识别生物素。因此，Biotin-(AC5)2-OSu 被用作 Biotin Labeling Kit-NH2 中的生物素标记剂。

Labeling Procedure for IgG1. Prepare 10 mM of the biotin labeling reagent using DMSO.2. Prepare 100 μl of 1 mg per ml IgG buffer solution (pH 7.5-8.5) that does not contain any large molecules with amine compounds.3. Add 1-5ml biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37ºC for 1 hour.4. Remove excess biotin labeling reagent using gel filtration or dialysis.5. Prepare solutions for further experiment using an appropriate buffer such as PBST (0.05% Tween 20/PBS).

1. J. Wormmeester, F. Stiekema and C. Groot, Immunoselective Cell Separation, Methods Enzymol., 1990, 184, 314.2. J. J. Leary, D. J. Brigati and D. C. Ward, Rapid and Sensitive Colorimetric Method for Visualizing Biotin-labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-blots, Proc. Batl. Acad. Sci. USA, 1983, 80, 4045.3. W. T. Lee and D. H. Conrad, The Murine Lymphocyte Receptor for IgE. II. Characterization of the Multivalent Nature of the B Lymphocyte Receptor for IgE, J. Exp. Med., 1984, 159, 1790.4. D. R. Gretch, M. Suter and M. F. Stinski, The use of Biotinylated Monoclonal Antibodies and Streptavidin Affinity Chromatography to Isolate Herpesvirus Hydrophobic Proteins or Glycoproteins, Anal. Biochem., 1987, 163, 270 .5. M. Shimkus, J. Levy and T.Herman, A Chemically Cleavable Biotinylated Nucleotide: Usefulness in the Recovery of Protein-DNA Complexes from Avidin Affinity Columns, Proc. Natl. Acad. Sci. USA, 1985, 82, 2593.6. W. J. LaRochelle and S. C. Froehner, Immunochemical Detection of Proteins Biotinylated on Nitrocellulose Replicas, J. Immunol. Methods, 1986, 92, 65.7. P. S. Anjaneyulu and J. V. Staros, Reactions of N-hydroxysulfosuccinimide Active Esters, Int. J. Pep. Protein Res., 1987, 30, 117.8. H. M. Ingalls, C. M. Goodloe-Holland and E. J. Luna, Junctional Plasma Membrane Domains Isolated from Aggregating Dictyostelium Discoideum Amebae, Proc. Natl. Acad. Sci. USA, 1986, 83, 4779.9. J. L. Guesdon, T. Ternynck and S. Avrameas, The Use of Avidin-biotin Interaction in Immunoenzymatic Techniques, J. Histochem, Cytochem, 1979, 27, 1131.

Dojindo,AB-NTA游离酸/100/A459,氨基丁基-NTA (AB-NTA) 游离酸替代 AB-NTA

氨基丁基-NTA (AB-NTA) 游离酸替代 AB-NTA（二钠盐形式；原产品代码：A296-10）。新的游离酸形式比二钠盐形式的吸湿性更小。在缓冲液中溶解新的酸形式与在缓冲液中溶解二钠盐的过程相同。新的酸形式也可以通过超声（最高 3%）溶解在水中。 Hochuli 博士于 1987 年首次使用氨基丁基-NTA 纯化重组蛋白（“Histag Etechnique”）。从那时起，AB=NTA 已成为将蛋白质以高特异性固定在玻璃或金电极等固体表面上的不可或缺的工具。固体表面通过 AB-NTA 修饰，并通过 Ni (II) 进行生物功能化，其中基因表达的蛋白质在其末端带有六组氨酸延伸。 His-tag 技术变得越来越重要，特别是在表面等离子共振和通过 X 射线干扰对蛋白质进行结构分析方面。使用 His-tag 技术，Noji 博士能够通过荧光显微镜直接观察到 F1-ATPase 的旋转。

Chemical Structure

1. E. Hochuli, H. Doeli and A. Schacher, New Metal Chelate Adsorbent Selective for Proteins and Peptides Containing Neighbouring Histidine Residues, J. Chromatogr., 1987, 411, 177.2. E. Hochuli, Large-scale Chromatography of Recombinant Proteins, J. Chromatogr., 1988, 444, 293.3. Y. C. Sasaki, Y. Suzuki and T. Ishibashi, Fluorescent X-ray Interference from a Protein Monolayer, Science, 1994, 263, 62.4. G. B. Sigal, C. Bamdad, A. Barberis, J. Strominger and G. M. Whitesides, A Self-assembled Monolayer for the Binding and Study of Histidine-tagged Proteins by Surface Plasmon Resonance, Anal. Chem., 1996, 68, 490.5. E. L. Schmid, T. A. Keller, Z. Dienes and H. Vogel, Reversible Oriented Surface Immobilization of Functional Proteins on Oxide Surface, Anal. Chem., 1997, 69, 1979.6. R. Yasuda, H. Noji, K. Kinosita, and M. Yoshida, F1-ATPase is a Highly Efficient Molecular Motor that Rotates with Discrete 120°Steps, Cell, 1998, 93, 1117.

Dojindo,DAOS/1/OC06

 1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982; 30:2492-2497.2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982; 136:121-127.3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984; 56:2849-2850.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,WST-1/500/W201,羟基来开发水溶性四唑盐 (WST)

Product Description of WSTs

通过在四唑盐的苯环上引入正电荷或负电荷和羟基来开发水溶性四唑盐 (WST)。 正电荷，例如三烷基铵基团，提高了甲臜染料的水溶性。 然而，大的阳离子很容易与有机阴离子如羧酸根或磷酸根一起沉淀出来。 虽然羟基也提高了四唑盐的水溶性，但相应的甲臜染料的水溶性不够。 Dojindo 的 WST 在苯环上直接或间接添加磺酸基团，以提高水溶性。 Dojindo 还提供了几种新开发的苯偶氮型四唑盐，它们很容易用 NADH 或其他还原剂还原，得到橙色或紫色的甲臜染料。 由于苯偶氮基，颜色随重金属离子而变化。 由于同仁堂 WST 的水溶性很高，因此可以制备 10 mM 至 100 mM 的溶液。

1. M. Ishiyama, et al., A New Sulfonated Tetrazolium Salt That produces a Highly Water-soluble Formazan Dye. Chem Pharm Bull. 1993;41:1118-1122.2. T. Yano, et al., Ras Oncogene Enhances the Production of a Recombinant Protein Regulated by the Cytomegalovirus Promoter in BHK-21 Cells. Cytotechnology. 1994;16:167-178.3. K. Teruya, et al., Ras Amplification in BHK-21 Cells Produces a Host Cell Line for Further Rapid Establishment of Recombiant Protein Hyper-producing Cell Lines. Biosci Biotech Biochem. 1995;59:341-344.4. M. Ishiyama, et al., Novel Disulfonated Tetrazolium Salt That can be Reduced to a Water-soluble Formazan and Its Application to the Assay of Lactate Dehydrogenase. Analyst. 1995;120:113-116.5. M. Ishiyama, et al., Novel Cell Proliferation and Cytotoxicity Assays Using a Tetrazolium Salt That Produces a Water-soluble Formazan Dye. In Vitro Toxicol. 1995;8:187-190.6. S. Q. Liu, et al., Induction of Human Autologous Cytotoxic T Lymphocytes on Formalin-Fixed and Paraffin-Embedded Tumour Sections. Nat Med. 1995;1:267-271.7. T. Takenouchi, et al., Trophic Effects of Substance P and β-Amyloid Peptide on Dibutyryl Cyclic AMP-Differentiated Human Leukemic (HL-60) Cells. Life Sci. 1995;56:PL479-PL484.8. T. Iwaki, et al., β-Crystallin in C6 Glioma Cells Supports their Survival in Elevated Extracellular K+: the Implication of a Protective Role of β-Crystallin Accumulation in Reactive Glia. Brain Res. 1995;673:47-52.9. M. Ishiyama, et al., A Combined Assay of Cell Viability and vitro Cytotoxycity with a Highly Water-soluble Tetrazolium Salt, Neutral Red and Crystal Violet. Biol Pharm Bul. 1996;19:1518-1520.10. T. Mosmann, et al., Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J Immunol Methods. 1983;65:55-63.11. A. H. Cory, et al., Use of an Aqueous Soluble Tetrazolium/Formazan Assay for Cell Growth Assays in Culture. Cancer Commun. 1991;3:207-212.

Dojindo,BAPTA-AM/25/B018,BAPTA 是由 Tsien 博士开发的钙选择性螯合剂

Product Description
BAPTA 是由 Tsien 博士开发的钙选择性螯合剂。 它的 logKCa=6.97 和 logKMg=1.77。 基本螯合单元类似于EGTA，但两个脂肪族氮原子被芳香族氮原子取代。 因此，BAPTA 在生理 pH 下不被质子化。 BAPTA 具有 pKa3=5.47 和 pKa4=6.36。 该性质表明去质子化步骤不包括在其钙络合步骤中，并且它具有比EGTA更高的络合率，因为它不受质子干扰的影响。 BAPTA-AM 是 BAPTA 的乙酰氧基甲酯衍生物，可以使用 AM 方法轻松加载到细胞中。 BAPTA-AM 可用于控制细胞内钙浓度。

Hydrolysis of AM ester

1. R. Y. Tsien, New Calcium Indicators and Buffers with High Selectivity against Magnesium and Protons: Design, Synthesis, and Properties of Prototype Structures. Biochemistry. 1980;19:2396-2404.2. R. Y. Tsien, A Non-disruptive Technique for Loading Calcium Buffers and Indicators into Cells. Nature. 1981;290:527-528.3. J. I. Korenbrot, et al., The Use of Tetracarboxylate Fluorescent Indicators in the Measurement and Control of Intracellular Free Calcium Ions. Soc Gen Physiol Ser. 1986;40:347-363.4. S. M. Harrison, et al., The Effect of Temperature and Ionic Strength on the Apparent Ca-affinity of EGTA and the Analogous Ca-chelators BAPTA and Dibromo-BAPTA. Biochim Biophys Acta. 1987;925:133-143.5. E. W. Gelfand, et al., Dissociation of Unidirectional Influx of External Ca2+ and Release from Internal Stores in Activated Human T Lymphocytes. Eur J Immunol. 1990;20:1237-1241.6. J. P. Kao, et al., Active Involvement of Ca2+ in Mitotic Progression of Swiss 3T3 Fibroblasts. J Cell Biol. 1990;111:183-196.7. M. L. Schubert, et al., Functionally Distinct Muscarinic Receptors on Gastric Somatostatin Cells. Am J Physiol. 1990;258:G982-G987.8. Y. Tojyo, et al., Inhibitory Effects of Loading with the Calcium-chelator 1, 2-Bis(o-aminophenoxy)ethane-N, N, N E N Etetraacetic acid (BAPTA) on Amylase Release and Cellular ATP Level in Rat Parotid Cells. Biochem Pharmacol. 1990;39:1775-1779.

Dojindo,生物素SAM形成试剂/1/B564

DescriptionManualS.D.S

Product Description

有三种方法可以将蛋白质固定在由自组装单层 (SAM) 组成的表面等离子共振 (SPR) 或石英晶体微天平 (QCM) 生物传感器上； 1) 胺官能团在蛋白质和活化的羧基-SAM上形成共价键； 2) 通过以 Ni-NTA 为末端的 SAM 通过 His-Tag 固定蛋白质； 3) 通过生物素和抗生物素蛋白相互作用固定蛋白质。 因为生物素-抗生物素蛋白的形成被称为一种简单的反应，所以它被广泛用于蛋白质的固定化（图 1）。

Dojindo,15-羧基-1-十五烷硫醇/10/C429,羧基烷硫醇用于修饰金表面以在其上引入羧基

羧基烷硫醇用于修饰金表面以在其上引入羧基。羧基通常转化为活化的 N-羟基琥珀酰亚胺酯，它与生物材料的胺基反应。 Dojindo 新开发的 15-Carboxy-1-pentadecanethiol 具有 15 个碳链，是市场上羧基烷硫醇中最长的烷硫醇。包括 Carboxy-EG6-十一烷硫醇在内的五种不同的羧基烷硫醇可用于金表面改性。 Malone 和其他人使用 15-Carboxy-1-pentadecanethiol 制造了一种高度灵敏的 SPR 传感器。 Glenn 和他的同事使用羧基烷硫醇和聚-L-赖氨酸来制造固定的细胞色素 b5 多层电极。 Mizutani 和其他人以类似的方式制造了固定化葡萄糖氧化酶多层电极。两组都报告了从生物材料到金表面的电子转移。这些类型的多层膜电极非常适用于扩散电子转移的研究。 Frisbie 和其他人开发了一种新方法，化学力显微镜，用于获得图案样品表面的粘合剂相互作用和摩擦图像。他们使用原子力显微镜 (AFM) 来测量化学上不同官能团的相互作用和空间映射。 Frisbie 和其他人在 AFM 悬臂尖端的金表面上形成了羧基烷硫醇单分子层。他们使用原子力显微镜测量分子修饰的探针尖端和有机单层之间的粘附力和摩擦力，这些有机单层以光刻定义的不同官能团图案终止。

1. M. R. Malone, J-F. Masson, S. Beaudoin, K. S. Booksh, Proceedings of SPIE-The International Society for Optical Engineering, 2005, 6007.

Dojindo,Biotin-PE-maleimide/10/B592,抗生物素蛋白-生物素系统免疫学和组织化学

抗生物素蛋白-生物素系统在免疫学和组织化学中有许多应用。抗生物素蛋白和生物素之间的相互作用非常强，解离常数约为 10-15 M。生物素通常添加到抗 IgG 和抗 IgM 等一抗或二抗中。在用生物素标记的抗体制备抗原-抗体复合物后，使用酶或荧光团标记的抗生物素蛋白或链霉抗生物素蛋白进行抗原的比色或荧光检测。马来酰亚胺生物素在 pH 7-7.5 下与硫醇化合物反应，例如具有巯基的蛋白质或肽。马来酰亚胺与巯基反应生成硫醚键。虽然其他马来酰亚胺生物素试剂必须溶解在 DMSO、DMF 或酒精中，但生物素-PE-马来酰亚胺可以溶解在 pH 7.4 的 PBS 中，以制备 2 mM 溶液，而无需使用有机溶剂。马来酰亚胺与巯基的反应性高于溴乙酰胺，因此马来酰亚胺生物素所需浓度远低于溴乙酰胺生物素。 Biotin-PEmaleimide 和 Biotin-PEAC5-maleimide 在 DMSO 中的储备溶液在 -20ºC 下可稳定保存一年。

Labeling Procedure for Reduced IgG1. Prepare 10 mM of the biotin labeling reagent using DMSO.2. Prepare 100 μl of 1 mg per ml reduced IgG/ml buffer solution that does not contain any large molecules with SH groups. Reduced IgG can be prepared by TCEP (tricarboxyethylphosphine), DTT, or 2-mercaptoethylamine.3. Add 1-5 μl of biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37ºC for 1 hour.4. Remove excess biotin labeling reagent using a gel column or a Filtration tube.5. Prepare solutions for further experiments using an appropriate buffer, such as PBST (0.05% Tween 20/PBS).

1. Hashida, M. Imagawa, S. Inoue, K. H. Ruan and E. Ishikawa , Mor Useful Maleimide Compounds for the Conjugation of Fab Eto Horseradish Peroxidase throuth Thiol Groups in the Hinge, J. Appl. Biochem., 1984, 6, 56.2. E. Ishikawa, M. Imagawa, S. Hashida, S. Toshitake, Y. Hamaguchi and T. Ueno, Enzyme-labeling of Antibodies and their Fragments for Enzyme Immunoassay and Immunohistochemical Staining, J. Immunoassay, 1983, 4, 209.3. H. -J. Friesen, P. Hermentin and P. Gronski, Novel Maleimido-Biotins for the Selective Biotinylation of Sulfhydrils, Protides Biol. Fluids, 1987, 34, 43.4. E. Ishikawa, S. Hashida, T. Kohno, T. Kotani and S. Ohtani, Modification of Monoclonal Antibodies with Enzymes, Biotin, and Fluorochromes and Their Applications, Immunol. Ser., 1987, 33, 113.5. R. B. del Rosalio and R. L. Wahl, Disulfide Bond-targeted Radiolabeling : Tumor Specificity of a Streptavidine-biotinylated Monoclonal Antibody Complex, Cancer Res.(Suppl.), 1990, 50, 804S.

Dojindo,8-氨基-1-辛硫醇盐酸盐/100/A424

氨基烷硫醇用于修饰金表面以在表面上引入氨基。 Dojindo 新开发的 16-Amino-1-hexadecanethiol 具有 16 个碳链，是市场上最长的烷硫醇。由于烷烃基团之间的范德华力较大，预计在氨基烷硫醇化合物中，16-氨基-1-十六烷硫醇将在金表面上形成最稳定的SAM。五种不同的氨基烷硫醇，包括氨基-EG6-十一烷硫醇、盐酸盐，可用于金表面改性。氨基-EG6-十一硫醇用于亲水表面制备。氨基通常使用胺反应性材料（例如蛋白质或生物材料）进行修饰，以使金表面功能化。一些研究人员已经报道了短烷基链氨基烷硫醇的 SAM，并且关于长烷基链化合物的报道越来越多。 Takahara 等人在金电极上形成了单层 11-Amino-1-十一硫醇，并使用伏安法研究了末端基团对二茂铁衍生物氧化还原反应的影响。他们还报道了氨基烷硫醇的烷基链长度与连接到末端氨基的 2,3-二氯-1,4-萘醌的氧化还原行为之间的关系。 Tanahashi 和同事用几种功能化烷硫醇的 SAM 修饰了金表面。他们使用 X 射线光电子能谱 (XPS) 测量和石英晶体微天平 (QCM) 方法报告了它们的末端官能团对模拟体液中磷灰石形成的影响。

1. J. M. Brockman, A. G. Frutos and R. M. Corn, A Multistep Chemical Modification Procedure To Create DNA Arrays on Gold Surface for the Study of Protein-DNA Interactions with Surface Plasmon Resonance Imaging, J. Am. Chem. Soc., 1999, 121, 8044.2. Y. Yoshimi, T. Matsuda, Y. Itoh, F. Ogata and T. Katsube, Surface Modifications of Functional Electrodes of a Light Addressable Potentiometric Sensor (LAPS): Non-Dependency of pH Sensitivity on the Surface Functional Group, Mater. Sci. Eng. C, 1997, 5, 131.3. M. Tanahashi and T. Matsuda, Surface Functional Group Dependence on Apatite Formation on Self-assembled Monolayers in a Simulated Body Fluid, J. Biomed. Mater. Res., 1997, 34, 305.4. J. Tien, A. Terfort and G. M. Whitesides, Microfaburication through Electrostatic Self-Assembly, Langmuir, 1997, 13, 5349.5. F. Mukae, H. Takemura and K. Takehara, Electrochemical Behavior of the Naphtoquinone Anchored onto a Gold Electrode through the Self-Assembled monolayers of Aminoalkanethiol, Bull. Chem. Soc. Jpn., 1996, 69, 2461.6. S. Rubin, G. Bar, R. W. Cutts, J. T. Chow, J. P. Ferraris, and T. A. Zawodzinski, Electrical Communication Between Glucose Oxidase and Different Ferrocenylalkanethiol Chain Lengths, Mater. Res. Soc. Symp. Proc., 1996, 413, 377.7. K. Takehara and H. Takemura, Electrochemical Behaviors of Ferrocene Derivatives at an Electrode Modified with Terminally Substituted Alkanethiol Monolayer Assemblies, Bull. Chem. Soc. Jpn., 1995, 68, 1289.8. K. Takehara, H. Takemura and Y. Ide, Electrochemical Studies of the Terminally Substituted Alkanethiol Monolayers Formed on a Gold Electrode; Effect of the Terminal Group on the Redox Responses of Fe(CN)63-,Ru(NH3)63+ and Ferrocenedimethanol, Electrochim. Acta, 1994, 39, 817.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,异硫氰酸苄基NTA/10/I279

异硫氰基苄基-NTA 用于修饰胺基连接的表面。 通过附着在表面上的 NTA 部分，基因表达的蛋白质在其末端带有六组氨酸延伸，可以通过 Ni (II) 固定（His-Tag 方法）。 使用这种技术，Noji 博士及其同事能够用荧光显微镜直接观察 F1-ATPase 的旋转。

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,NAD/NADH Assay Kit-WST/100/N509,NAD/NADH 检测试剂盒

使用该试剂盒中的提取缓冲液和过滤管，可以通过脱蛋白轻松制备来自细胞培养物的细胞裂解物。 细胞内 NADH 水平可以通过细胞裂解物的热处理来量化。 此外，可以通过从独立测量的总 NAD+/NADH 水平中减去 NADH 水平来确定细胞内 NAD+ 水平。

PrincipleNAD/NADH Assay Kit-WST 能够量化细胞中总 NAD+/NADH、NADH 和 NAD+ 的量并测量它们的比率。

Measurement of NAD+ and NADH

Cell lysate from a cell culture can be easily prepared via Deproteinzation using the extraction buffer and filtration tubes found within this kit. Intracellular NADH levels can be quantified by heat treatment of cell lysate. Additionally, intracellular NAD+levels can be determined by subtracting NADH levels from independently measured total NAD+/NADH levels.

Study of NAD+/NADH as MarkersRecently, it has become clear that Sirtuin is linked to longevity and plays a role in NAD+ level regulation. Also Sirtuin has been recognized as a marker necessary to understanding biological states, such as obesity & diabetes, as well as cellular differentiation.

In the experiment shown below, the NAD+ /NADH levels & ratio were determined using HeLa cells.

Standard curves were constructed using different concentrations of HeLa cells (2.5×105 and 5.0×105 cells) cultured in growth media. The standard curves were then used to determine the intracellular NAD+ and NADH levels. As a result, NAD+ and NADH levels varied depending on cell number while the change in cell number had no effect on the NAD+/NADH ratio.

Measurement of NAD+/NADH in Combination with Lactate Assay KitChange in metabolic activity was observed when the glycolytic inhibitor 2-Deoxy-D-glucose was added to HeLa cells.

2-Deoxy-D-glucose was added to HeLa cells (1×106 cells) to obtain a final concentration of 6 mmol/l 2-Deoxy-D-glucose. After 24 hours of incubation, lactate levels in the supernatant were quantified using the Lactate Assay Kit-WST (Item#: L256), and the NAD+/NADH ratio was determined with the cell pellet after removing the supernatant using the NAD/NADH Assay Kit-WST.

As a result, intracellular glycolysis was inhibited by 2-Deoxy-D-glucose, which led to decreased lactate levels and an increase in the NAD+/NADH ratio.

1. C. Henninger and G. Fritz, “Statins in anthracycline-induced cardiotoxicity: Rac and Rho, and the heartbreakers.”, Cell Death Dis. 2017, 8(1), e2564.2. S. Mandziuk, R. Gieroba, A. Korga, W. Matysiak, B. Jodlowska-Jedrych, F. Burdan, E. Poleszak, M. Kowalczyk, L. Grzycka-Kowalczyk, E. Korobowicz, A. Jozefczyk and J. Dudka, “The differential effects of green tea on dose-dependent doxorubicin toxicity.”, Food Nutr Res., 2015, 59, 29754.

How many samples can I measure?

The number of samples that can be measured when the standard sample is serially diluted from 2 μmol / l is shown in the table above.* The number of samples that can be recorded when the sample is measured in triplicates.It is necessary to create a calibration curve per measurement when you perform the measurement separately. If the measurement is done separately, the above sample number will be less.

I do not have a 450 nm filter. What other filters can I use?
You can use filters with an absorbance between 450 nm and 490 nm. However, the absorbance value can become lower if the sample is not measured at 450 nm.
Can I purchase the filtration tube separately?

No, we don’t sell the filtration tube included in the kit separately. If you need additional supplies, you can also use a commercially available filtration tube.Supplier：Pall CorporationProduct：Nanosep® MF Centrifugal Devices (MWCO：10K, color：blue)

How stable is Working solution?
You can’t store the Working solution. Please prepare the Working solution prior to use. Please protect from light because the Working solution ins light-sensitive. The Working solution is stable for 4 hours at room temperature with protection from light.
Our samples did not change in color, are there any reasons for this?
NAD level contained in the sample may be lower than the detection limit that can be measured using this kit.Please increase the number of cells or lower the dilution ratio if you dilute the sample.

Dojindo,葡萄糖分析试剂盒WST/200/G264

原理本试剂盒可以通过测量有色WST甲臜染料的吸光度来检测细胞培养基中的葡萄糖或细胞内的葡萄糖。 吸光度取决于样品中存在的葡萄糖量。该试剂盒包含可用于创建标准曲线的葡萄糖标准溶液。 这允许对样品中存在的葡萄糖水平进行定量。

程序 程序非常简单，您只需在添加培养上清液或组织/细胞裂解物后，在添加试剂之前孵育板即可。

标准曲线的制备 样品中的葡萄糖水平可以通过使用该试剂盒中包含的葡萄糖标准建立的校准曲线来测量。 如果葡萄糖水平大于或等于 0.5 mmol/L，则必须在测量前稀释样品。

结合 Lactate Assay Kit 测量葡萄糖水平通过使用 Glucose Assay Kit-WST 和 Lactate Assay Kit-WST，我们成功测量了将蛋白转运抑制剂根皮素添加到 Jurkat 细胞中时的代谢活性变化。

Precautions when using this kit.

Cell counts may need to be normalized. When cells are analyzed in a microplate, the results obtained may sometimes differ depending on cell numbers per well.In such cases, normalization of the measured values obtained from cell counting and total protein will be necessary.In this kit, cell numbers can be easily measured by the fluorescence intensity induced by a reagent added to cell culture medium for staining nuclei.

I do not have a 450 nm filter. What other filters can I use?
You can use filters with an absorbance between 450 nm and 490 nm. However, the absorbance value can become lower if the sample is not measured at 450 nm.
Can I quantify L-Glucose levels?
No, the kit is used for the quantification of β-D-Glucose levels.
How many samples can I measure?
* The number of samples that can be recorded when the standard curve and sample is measured in triplicates.*A plate layout for Glucose standard solution and sample

Dojindo,Cell Counting Kit-F/500/CK06,细胞计数试剂盒

Cell Counting Kit-F(CCK-F) 用于活细胞数量的荧光测定。 细胞中被酯酶水解的荧光染料钙黄绿素的量与培养基中活细胞的数量成正比（图 1）。 由于培养基中的酯酶和酚红会干扰荧光测量，因此在添加 Calcein-AM 测定溶液之前，必须用 PBS 替换细胞培养基。 钙黄绿素的激发和发射波长分别为 485 nm 和 535 nm（图 3）。 10 到 30 分钟的孵育为细胞活力测定提供了足够的荧光强度。
使用标准 96 孔板时，每孔（100 μl 培养基）至少需要 50 个细胞。但是，我们建议每孔至少使用 1,000 个细胞，以获得更可靠和一致的数据。
钙黄绿素会染色活细胞吗？
是的，钙黄绿素留在细胞内。可以使用荧光显微镜观察活细胞。如果需要更长的观察期，请尝试 CFSE（产品代码：C375-10）。

酚红会影响检测吗？
是的。在向板中添加分析溶液之前需要进行洗涤过程。只有 PBS 的孔也有相当高的荧光背景，因此有必要从总荧光中减去背景荧光。

我可以使用该试剂盒进行细菌细胞计数吗？
不可以。Calcein-AM 不能穿过细菌细胞壁，因此无法使用该试剂盒对它们进行染色。

CCK-F 检测溶液稳定吗？
不会。CCK-F 中的 Calcein-AM 在 PBS 中很容易水解。仅为实验准备所需体积的测定溶液。

CCK-F 与胸苷掺入测定之间是否存在相关性？
是的。但是，请注意，由于 CCK-F 使用与胸苷检测不同的检测机制，结果可能会有所不同。比较数据显示在技术手册中，该手册可从 www.dojindo.com/tm 获得。

CCK-F 检测溶液对细胞有毒吗？
由于检测溶液是用 PBS 制备的，因此某些细胞可能会受到 PBS 的影响。此外，钙黄绿素与细胞中的钙离子结合，因此游离钙离子的减少会对细胞功能造成一些损害。

我还可以使用哪些其他过滤器？
您可以使用 460 nm 和 490 nm 之间的激发滤光片和 510 nm 和 540 nm 之间的发射滤光片。

我可以将 CCK-F 用于 384 孔板吗？
通过将 5 μl（而不是 10 μl）CCK-F 测定溶液添加到每孔 50 μl PBS 溶液中，CCK-F 可用于 384 孔板。

Dojindo,HilyMax/1/H357,A549 细胞的信号转导通过 TNF-α 刺激得到证实

已经开发了多种方法来在哺乳动物细胞中表达特定蛋白质。将 DNA 引入细胞的第一种方法是磷酸钙沉淀。然而，转染效率很差，细胞间变异率很高。介绍的第二种方法是 DEAEsephadex 方法。转染效率显着提高，但该方法仍不能用于所有细胞，需要重金属离子来提高转染效率。随后开发了阳离子脂质体方法，该方法被证明是一种将 DNA 和 RNA 转染到细胞中的更好方法。使用的其他方法是磁珠、金属粒子射击和电穿孔。然而，阳离子脂质体法不需要任何特殊仪器或特殊技能。因此，许多研究人员都在使用这种方法。 HilyMax 是一种新开发的基因转染试剂，可形成脂质体，用于高效基因转染多种细胞。此外，在信号转导研究中，由于引入细胞内的试剂不会中断细胞内信号通路，HilyMax 能提供更好的信号（图 3）。由于生长培养基中的血清不会干扰使用 HilyMax 的转染，因此在转染过程中无需更换培养基。 HilyMax 不含可能干扰转染的生物成分。

Principle

HilyMax 很容易与 DNA 相互作用，因为阳离子脂质体 (+) 和阴离子 DNA(-) 自发形成 DNA 脂质体复合物。 DNA-HilyMax 复合物的总电荷为正电荷，因此 DNA-HilyMax 复合物静电结合在阴离子细胞表面，并通过内吞作用将 DNA 引入细胞。

Procedure

The procedure is extremely simple. No exchange of the media is required during the whole process,because serum in the medium does not interfere with the transfection.

Cell Signaling ResearchFigure 1. Suitable signal transduction research using HilyMax.

来自 A549 细胞的信号转导通过 TNF-α 刺激得到证实。 为了检测细胞反应，用 HilyMax 或 L2000 转染 IL-8 依赖性荧光素酶表达载体。 信号转导反应在刺激和抑制后检测为荧光素酶活性。 使用 HilyMax 的信号响应与受刺激细胞中表达的 IL-8 的量有关。如果您对此问题感兴趣，请单击此处获取更多信息。

Comparison Data in Insect Cell<New Data>

Data was kindly provided by Dr. Takashi Suzuki at Max Planck Institute of Neurobiology.

Culture ConditionCell: S2(Schneider 2) cell, 200,000 cells/wellMedia: Schneider’s Drosophila medium with 10% FCSAntibiotics: 50 units Penicillin/ml, 20 µg Streptomycin/mlMicroplate: 24-well plate

Transfection ConditionVector: 1 µg/well <pAct Gal4(6 kb), pUAS-mCD8::GFP(10 kb)>Reagent: 5 µl/well <HilyMax or Cfectin>*Medium was changed in 4 hours after transfection.*Schneider’s Drosophila medium(without seum and antibiotics) was used for the complex preparation.

GFP Transfected Cells with HilyMax

Transfection Efficiency in Various Cell Lines

Comparison Data of Transfection Efficiency with Commercially Available Reagents

The tranfection efficiency of HilyMax is higher than commercially available reagents in widely used cells. GFP expressed DNA was transfected using HilyMax and the other transfection reagents in serum containing medium. The amount of expressed protein indicates the transfection efficiency.

Transfection efficiency is low.

1. The volume of HilyMax is not enough. Increase the HilyMax volume.2. Cell density is too high. Reduce the cell density. The appropriate cell density for transfection is about 40-90%.3. HilyMax reagent is not be dissolved completely. Please check that the HilyMax solution is homogeneous.4. Incubation time for the preparation of HilyMax and DNA complex is too long.5. Your culture medium for DNA-HilyMax complex formation contains serum and/or antibiotics. Please use serum-free and antibiotics-free medium for the complex formation.

Toxicity is high. Most cells died.

1. Reduce the amount of DNA and/or HilyMax. Prepare the complex.2. Cell density is too low. Reduce the cell density. The appropriate cell density for transfection is about 40-90%.

Dojindo,8-Hydroxy-1-octanethiol/10/H338

羟基烷硫醇用作金表面上的稀释试剂以控制反应基团的密度，或用作封闭剂以防止分析物在表面上的非特异性结合。新开发的 16-Hydroxy-1-hexadecanethiol 具有 16 个碳链，是市场上羟基烷硫醇中最长的烷硫醇。总共有 6 种不同的羟基烷硫醇，包括可用于金表面改性的羟基-EG6-十一烷硫醇和羟基-EG3-十一烷硫醇。当应用 16-Amino-1-hexadecanethiol 或 15-Carboxy-1-pentadecanethiol 时，16-Hydroxy-1-hexadecanethiol 用于制备均匀且高度定向的 SAM。 Herne 和他的同事在金表面上制造了硫醇衍生的单链 DNA (HS-ss-DNA) 和 6-Hydroxy-1-己硫醇的混合 SAM，以防止 HS-ss-DNA 的非特异性吸附。 Perez-Luna 和其他人在金表面上制作了生物素末端硫醇和 11-羟基-1-十一烷硫醇的混合 SAM。它们阻止了野生型链霉亲和素和链霉亲和素突变体的非特异性吸附。 Dubrovsky 及其同事使用 11-Hydroxy-1-十一硫醇控制了蛋白质在镀金硅胶表面上的非特异性吸附。他们提到了镀金硅胶在制备用于生物测定的明确的、表面功能化的支持物方面的有用性。

Dojindo,ARP醛反应探针/25/A305,醛反应探针 (ARP) 因其醛特异性反应性

醛反应探针 (ARP) 因其醛特异性反应性而被用于检测 DNA 中的脱碱基位点（AP 位点、脱嘌呤/脱嘧啶位点）。 ARP 与醛基和酮基反应，并将生物素添加到 DNA 中的 AP 位点。 然后使用过氧化物酶标记的抗生物素蛋白和氧化显色染料检测生物素标记的 DNA。 ARP 方法已被用于检测 1×104 个 DNA 核苷酸中少于 1 个 AP 位点。 Dojindo 提供 DNA 损伤定量试剂盒（产品代码：DK02），其中包含确定每 1×105 个碱基对中 1 到 40 个 ARP 位点所需的所有试剂和组分。 ARP 极易溶于水，储备液可在 4ºC 下储存一年而不会显着降低反应性。

Labeling Procedure for DNA Abasic Site1. Prepare 10 mM ARP with water.2. Adjust the DNA concentration to 100 μg per ml with TE buffer (pH 7.4).3. Mix equal volumes of the DNA and ARP solutions and incubate at 37ºC for 1 hour.4. Isolate the ARP-labeled DNA using either ethanol precipitation or membrane filtration tube.5. Dissolve the DNA pellet in TE to prepare 10-100 μg per ml solution.

1. R. Rago, J. Mitchen and G. Wilding, DNA Fluorometric Assay in 96-well Tissue Culture Plates Using Hoechst 33258 after Cell Lysis by Freezing in Distilled Water, Anal. Biochem., 1990, 191, 31.2. K. Kubo, H. Ide, S. S. Wallace and Y. W. Kow, A Novel, Sensitive, and Specific Assay for Abasic Sites, The Most Commonly Produced DNA Lesion, Biochemistry, 1992, 31, 3703.3. H. Ide, K. Akamatsu, Y. Kimura, K. Michiue, K. Makino, A. Asaeda, Y. Takamori and K. Kubo, Synthesis and Damage Specificity of a Novel Probe for the Detection of Abasic Sites in DNA, Biochemistry, 1993, 32, 8276.4. M. Yao and Y. W. Kow, Strand-specific Cleavage of Mismatch-containig DNA by Deoxyinosine 3 EEndonuclease from Escherichia coli, J. Biol. Chem., 1994, 269, 31390-6.5. T. Shida, M. Noda and J. Sekiguchi, The recognition of DNA containning AP-site by E. coli endonuclease IV(exonuclease), Nucleic Acids Symposium Series, 1995, 34, 87.6. H. B. Sun, L. Qian and H. Yokota, Detection of abasic sites on individual DNA molecules using atomic force microscopy, Anal. Chem., 2001, 73, 2229.7. J. Nakamura, J. A. Swenberg, Endogenous Apurinic/apyrimidinic Sites in Genomic DNA of Mammalian Tissues, Cancer Res., 1999, 59, 2522-2526.8. Y. W. Kow and A. Dare, Detection of Abasic Sites and Oxidative DNA Base Damage using an ELISA-like Assay, METHODS, 2000, 22, 164.9. L. Yan, A. Bulgar, Y. Miao, V. Mahajan, J. R. Donze, S. L. Gerson and L. Liu, Combined Treatment with Temozolomide and Methoxyamine: Blocking Apurininc/Pyrimidinic Site Repair Coupled with Targeting Topoisomerase II, Clin. Cancer Res., 2007, 13, 1532.10. P. D. Chastain, II, J. Nakamura, J. Swenberg and D. Kaufman, Nonrandom AP site distribution in highly proliferative cells, FASEB J., 2006, 20, 2612.

Dojindo,BS3/50/B574,BS3是使氨基相互反应的交联剂

BS3是使氨基相互反应的交联剂。 由于BS3分子两端具有N-羟基琥珀酰亚胺活性酯，可选择性地与氨基反应，因此可以将载体蛋白与简单的半抗原或制备标记酶结合。 BS3 的化学结构具有连接部分的烷基链。 同时，DTSSP 和 DSP 能够切割容易被还原剂还原的引入二硫键的接头位点。 此外，由于具有磺酸基的活性酯基被引入到DTSSP和BS3中，因此可以在不使用有机溶剂如DMSO或DMF的情况下进行标记反应。

Homo-bifunctional Reagents

Product Name Code Length (Å)
BS3 B574 8.9
DTSSP D630 8.5

1. J.V. Staros, “N-Hydroxysulfosuccinimide Active Ester: Bis(N-hydroxysulfosuccinimide)Ester of Two Dicarboxylic Acids Are Hydrophilic, Membrane-impermeant, ProteinCross-Linkers”, Biochemistry, 1982, 21, 3950.2. J. M. Salhany, R. L. Sloan and K. A. Cordes, “In SituCross-linking of Human Erythrocyte Band 3 byBis8sulfosuccinimidyl)suberate”, J. Biol. Chem., 1990, 265(29),17688.3. J. E. Gestwicki, C. W. Cairo, L. E. Strong, K. A. Oetjen and L. L.Kiessling, “Influencing Receptor-Ligand Binding Mechanisms withMultivalent Ligand Architecture”, J. Am. Chem. Soc., 2002, 124,14922.4. S. S. Mark, N. Sandhyarani, C. Zhu, C. Campagnolo and C. A. Batt,”Dendrimer-functionalized Self-assembled Monolayers as a Surface PlasmonResonance Sensor Surface”, Langmuir, 2004, 20, 6808.

Dojindo,HDAOS/1/OC08

1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982;30:2492-2497.2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982;136:121-127.3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984;56:2849-2850.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,Sulfo EMCS/50/S024

杂双功能交联剂具有活化的酯和马来酰亚胺反应基团。 这些官能团分别与蛋白质的胺和巯基反应。 酶标记的半抗原是使用异双功能交联剂（例如 EMCS 或 GMBS）制备的。 交联反应需要中性 pH 和温和的温度，因为在交联反应中需要保持酶活性和抗体滴度。 可使用具有 3、5、7 或 10 个线性碳链的异双功能交联剂。 这些线性脂肪链充当两个反应位点与其水溶性试剂之间的间隔物。 在更宽的 pH 范围内，它们比芳香族交联剂（例如 succinimidyl-4-N-maleimidobenzoate）更稳定。

Conjugation of Macromolecules with Hetero-Bifunctional Cross-Linking Reagent

Hetero-bifunctional Reagents

Product Name Code Length (Å)
Sulfo-EMCS S024 9.4
Sulfo-GMBS S025 6.9
Sulfo-HMCS S026 13.0
Sulfo-KMUS S250 16.7
Sulfo-SMCC S330 8.0
1. J. V. Staros, N-Hydroxysulfosuccinimide Active Esters:Bis(N-hydroxysulfosuccinimide) Esters of Two Dicarboxylic acids Are Hydrophilic, Membraneimpermeant, Protein Cross-linkers, Biochemistry, 1982, 21, 3950.2. P. S. R. Anjaneyulu, James V. Staros, Reactions of N-Hydroxysulfosuccinimide Active Esters, Int. J. Peptide Protein Res., 1987, 30, 117.3. Y. Fukami, K. Sato, K. Ikeda, K. Kamisango, K. Koizumi and T. Matsuno, Evidence for Autoinhibitory Regulation of the c-src Gene Product a Possible Interaction Between the Src Homology 2 Domain and Autophosphorylation Site, J. Biol. Chem., 1993, 268, 1132.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,Biotin-AC5-OSu/10/B305

Product Description of Amine-Reactive Biotins抗生物素蛋白-生物素系统在免疫学和组织化学中有许多应用。抗生物素蛋白和生物素之间的相互作用非常强，解离常数约为 10-15 M。生物素通常添加到一抗或二抗中，例如抗 IgG 和抗 IgM。在用生物素标记的抗体制备抗原-抗体复合物后，使用酶或荧光素标记的抗生物素蛋白或链霉抗生物素蛋白对抗原进行比色或荧光检测。琥珀酰亚胺酯生物素在 pH 7-9 下与伯胺和仲胺（例如氨基酸和蛋白质）反应。琥珀酰亚胺酯与游离胺基反应生成稳定的酰胺键。琥珀酰亚胺基生物素试剂必须溶解在 DMSO、DMF 或酒精中。用 DMSO 制备的储备溶液在 -20ºC 下可稳定数月。磺基琥珀酰亚胺生物素试剂可溶于水，因此无需使用 DMF 或 DMSO 等有机溶剂。使用具有较长间隔物的生物素（如 Biotin-(AC5)2-OSu 或 Biotin-(AC5)2-Sulfo-OSu）制备的 IgG 具有更好的信噪比。较长的间隔使链霉亲和素或抗生物素 IgG 能够在没有结构抑制的情况下识别生物素。因此，Biotin-(AC5)2-OSu 被用作 Biotin Labeling Kit-NH2 中的生物素标记剂。

Labeling Procedure for IgG1. Prepare 10 mM of the biotin labeling reagent using DMSO.2. Prepare 100 μl of 1 mg per ml IgG buffer solution (pH 7.5-8.5) that does not contain any large molecules with amine compounds.3. Add 1-5ml biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37ºC for 1 hour.4. Remove excess biotin labeling reagent using gel filtration or dialysis.5. Prepare solutions for further experiment using an appropriate buffer such as PBST (0.05% Tween 20/PBS).

1. P. Kongtawelert and P. Ghosh, A New Sandwich-ELISA Method for the Determination of Keratan Sulphate Peptides in Biological Fluids Employing a Monoclonal Antibody and Labelled Avidin Biotin Technique., Clin. Chem. Acta,, 1990, 195, 17 .2. G. Paganelli, S. Pervez, A. G. Siccardi, G. Rowlinson, G. Deleide, F. Chiolerio, M. Malcovati, G. A. Scassllati and A. A. Epenetos, Intraperitoneal Radio-localization of Tumors Pre-targeted by Biotinylated Monoclonal Antibodies, Int. J. Cancer, 1990, 45, 1184.3. C. Wagener, U. Kruger and J. E. Shively, Selective Precipitation of Biotin-labeled Antigens or Monoclonal Antibodies by Avidin for Determining Epitope Specificities and Affinities in Solution-phase Assays, Methods Enzymol., 1990, 184, 518 .4. D. M. Boorsma, J. Van Bommel and E. M. Vander Raaij-Helmer, Simultaneous Immunoenzyme Double Labelling Using Two Different Enzymes Linked Directly to Monoclonal Antibodies or with Biotin-avidin, J. Microscopy, 1986, 143, 197.5. A. Komura, T. Tokuhisa, T. Nakagawa, A. Sasase, M. chihashi, S. Ferrone and Y.Mishima, Specific Killing of Human Melanoma Cells with an Efficient 10B-compound on Monoclonal Antibodies, Pigment Cell Res., 1989, 2, 259.6. R. Rappuoli, P. Leoncini, P. Tarli and P. Neri, Competitive Enzyme Immunoassay for Human Chorionic Somatomammotropin Using the Avidin-biotin System, Anal. Biochem., 1981, 118, 168.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,Allophycocyanin Labeling Kit-SH/3/LK24,别藻蓝蛋白标记试剂盒

藻胆蛋白是源自蓝细菌和真核藻类的荧光蛋白。它们的荧光远高于荧光素和罗丹明等化学荧光探针。别藻蓝蛋白 (APC) 是一种藻胆蛋白，在 660 nm 附近有红色荧光（图 1）。由于这种高荧光，藻胆蛋白标记的抗体和其他分子可以在流式细胞术和免疫染色中提供更高的灵敏度。别藻蓝蛋白标记试剂盒-SH 用于简单快速地制备 APC 标记的 IgG（图 2）。 SH-reactive APC（本试剂盒的一个组成部分）具有马来酰亚胺基团，无需任何活化过程即可轻松与目标分子的巯基形成共价键。该套件中的过滤管允许快速更换缓冲液并浓缩样品 IgG 溶液。该试剂盒包含 APC 标记所需的所有试剂，包括用于制备具有 SH 基团的还原型 IgG 的还原剂和用于偶联物的储存缓冲液。

Precaution♦ 使用本试剂盒标记的还原蛋白的分子量应大于 50,000。♦ 标记过程中，IgG 或别藻蓝蛋白结合的 IgG 始终在过滤管的膜上。♦ 如果 IgG 溶液中含有其他具有分子 重量大于 10,000，例如 BSA 或明胶，在使用该试剂盒标记别藻蓝蛋白之前纯化 IgG 溶液。 IgG 溶液可通过 IgG 纯化试剂盒（本试剂盒未提供）进行纯化。♦ 如果 IgG 溶液含有少量不溶性物质，则将溶液离心并使用上清液进行标记。

Can I use this kit for F(ab)”2?
Yes, please follow the labeling protocol for IgG. The recovery of the conjugate should be over 80%.
Can I use this kit for other proteins?
Yes, if the molecular weight of the reduced form is greater than 50,000 and it has a reactive SH group, or a disulfide group that can be reduced without losing activity. Follow the labeling protocol for IgG, and use 0.5-1 nmol of sample protein.
How many R-PE molecules per reduced IgG are introduced?
The average number of APC molecule per reduced IgG is 1 to 2.
Do I have to use the Filtration tube prior to labeling the protein?
If the protein solution does not contain small molecules with reactive SH groups and the concentration of the protein is 10 mg per ml, or about 70 μM, there is no need to use the Filtration tube. Just mix 10 μl of the sample solution with Reaction buffer and add the mixture to a vial of the SH-reactive R-PE.
Do I have to use WS buffer included with the kit?
Yes. Use the WS buffer to prepare a stock solution of the conjugate. However, you can choose any kind of buffer appropriate to dilute the conjugate stock solution for your experiment.

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,Nitro-TB/1/N011

Nitro-TB 用热水或热甲醇溶解。 其他有机溶剂如丙酮和乙醚不能溶解 Nitro-TB。 Nitro-TB 很容易被脱氢酶还原为紫色甲臜染料聚集体。 它用于琼脂糖凝胶上的脱氢酶检测。
1. K. Tsou 等人，一些对硝基苯基取代的四唑盐电子受体的合成，用于证明脱氢酶。 J Am Chem Soc. 1956；78:6139-6144.2。 J. R. Baker 等人，果糖胺测定的机制：反对超氧化物作为中间体在 Nitroblue Tetrazolium 还原中的作用的证据。 临床化学。 1993；39:2460-2465。

Dojindo,WST-9/100/W217

 Product Description of WSTs通过在四唑盐的苯环上引入正电荷或负电荷和羟基来开发水溶性四唑盐 (WST)。 正电荷，例如三烷基铵基团，提高了甲臜染料的水溶性。 然而，大的阳离子很容易与有机阴离子如羧酸根或磷酸根一起沉淀出来。 虽然羟基也提高了四唑盐的水溶性，但相应的甲臜染料的水溶性不足。 Dojindo 的 WST 在苯环上直接或间接添加磺酸基团，以提高水溶性。 Dojindo 还提供了几种新开发的苯偶氮型四唑盐，它们很容易用 NADH 或其他还原剂还原，得到橙色或紫色的甲臜染料。 由于苯偶氮基，颜色随重金属离子而变化。 由于 Dojindo 的 WST 的水溶性很高（WST-10 除外），因此可以制备 10 mM 至 100 mM 的溶液。

WST-1: M. Ishiyama, et al., Chem. Pharm. Bull, 41, 1118 (1993),M. Ishiyama, et al., In Vitro Toxicol, 8, 187 (1995),S. Shirahata, et al., Biosci. Biotech. Biochem., 59, 345 (1995),K. Teruya, et al., Biosci. Biotech. Biochem., 59, 341 (1995),Y. Kayamori, et al., Clin. Biochem., 30, 595 (1997);W. Zhang, et al., FASEB J., 20, 2496 (2006);S. Yang, et al., J. Pharmacol. Exp. Ther., 319, 595 (2006);D. Inokuma, et al., Stem Cells, 24, 2810 (2006);T. Ishikawa, et al., Arterioscler. Thromb. Vasc. Biol., 26, 1998 (2006).

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,羟基-EG3-十一烷硫醇/100/H354

聚乙二醇 (PEG) 广泛用于材料改性以提高表面的亲水性。 PEG 涂层材料通常在生理条件下更稳定。由于羟基-EGn-十一硫醇具有 3 或 6 个乙二醇单元、11 个碳原子和末端的 SH 基团，因此可用于在金表面制备高度定向和亲水性的 SAM，适用于生物材料标记表面。这是由于亲水性的提高。亲水表面可以防止蛋白质或其他生物材料发生非特异性结合。因此，该试剂制备的SAM将为开发生物材料传感器或DNA/蛋白质微阵列提供更好的表面。羟基-EGn-十一硫醇（n = 3, 6）用于根据引入到表面上的分子的密度来稀释羧基-SAM或氨基-SAM。有几篇关于标记蛋白质（如卵清蛋白和细胞色素 C）的论文。
C. Pale-Grosdemange 等人，肛门。 Chem., 71, 777 (1999);X.钱，等人，肛门。 Chem., 74, 1805 (2002);M.京等人，肛门。 Chem., 77, 7115 (2005);A. Subramanian 等人，生物传感器和生物电子学，21, 998 (2006)。

Dojindo,TODB/1/OC22号

1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982; 30:2492-2497.2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982; 136:121-127.3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984; 56:2849-2850.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,ADOS/1/OC01,Dojindo细胞分析

 1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982;30:2492-2497.2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982;136:121-127.3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984;56:2849-2850.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,Mitophagy Detection Kit/Mtphagy/MD01,线粒体自噬检测试剂盒

Mitophagy 检测试剂盒针对哺乳动物细胞进行了优化。
线粒体是细胞质细胞器之一，在细胞中起着至关重要的作用，例如产生细胞活力的能量。最近，线粒体自噬似乎与去极化线粒体的积累诱导的阿尔茨海默病和帕金森病有关。线粒体自噬作为一种特异性消除系统，将由氧化应激和DNA损伤引起的功能失调的线粒体隔离到自噬体中，与溶酶体融合并通过消化降解。该试剂盒由Mtphagy Dye、线粒体自噬检测试剂和Lyso Dye组成。 Mtphagy Dye 积聚在完整的线粒体中，通过化学键固定在其上，并因周围条件的影响而呈现出微弱的荧光。当 Mitophagy 被诱导时，受损的线粒体与溶酶体融合，然后 Mtphagy Dye 发出高荧光。为了确认 Mtphagy Dye 标记的线粒体和溶酶体的融合，可以使用本试剂盒中包含的 Lyso Dye。E. Fang 等利用 mt-mKeima 检测了番茄碱诱导的 HeLa 细胞线粒体自噬。使用我们的 Mitophagy Detection Kit 3 在原代大鼠皮层神经元和人类 SH-SY5Y 神经细胞中也检测到 Mitophagy。如果蛋白质表达/转染不适合实验，Mitophagy Detection Kit 将是一种有效的替代方法。有关 Mtphagy Dye 组合物和示例的更多信息，请参阅以下出版物：Iwashita H、Torii S、Nagahora N、Ishiyama M、Shioji K、Sasamoto K、Shimizu S、Okuma K。新型荧光小分子。“ACS Chem Biol, 2017, doi: 10.1021/acschembio.7b00647。注：Mtphagy Dye 和 Lyso Dye 正在申请专利。

Simple Procedure

CCCP（羰基氰化物间氯苯腙）已添加到正常和 Parkin 表达的细胞中。在正常HeLa细胞(A)(B)中未观察到强荧光。另一方面，在加入CCCP(C)后18小时，Parkin表达的细胞中显示出强荧光。一些斑点与自噬标记 (GFP-LC3) 共定位。此外，当自噬抑制剂巴弗洛霉素添加到停车表达的细胞中时，观察到 Mtphagy 染料的荧光受到抑制（D）。因为添加巴弗洛霉素会增加溶酶体的 pH 值。

饥饿细胞中的线粒体自噬检测
在线粒体自噬诱导条件下，活的 HeLa 细胞与 Mtphagy Dye 和 Lyso Dye 共染色。将 Mtphagy Dye 添加到 HeLa 细胞中，细胞在饥饿条件下孵育 6 小时。在观察细胞之前，将溶酶体染色染料 Lyso Dye 添加到 HeLa 细胞中。 Mtphagy Dye 的荧光强度在饥饿的 HeLa 细胞中增加，但在正常细胞中不增加。此外，Mtphagy Dye 与 Lyso Dye 在饥饿细胞中共同定位。

实验示例：CCCP 诱导线粒体自噬
羰基氰化物间氯苯腙 (CCCP) 作为线粒体解偶联试剂与 Parkin 表达的 HeLa 细胞诱导线粒体自噬

将 HeLa 细胞接种在 μ-slide 8 孔 (Ibidi) 上，并在 5%-CO2 培养箱中于 37oC 培养过夜。细胞通过HilyMax转染试剂（代码#：H357）转染Parkin质粒载体，37℃孵育过夜。 Parkin 表达的 HeLa 细胞用 Hanks 的 HEPES 缓冲液洗涤两次，然后在 37oC 下与 250 μl 含有 100 nmol/l MitoBright Deep Red（代码#：MT08）的 100 nmol/l Mtphagy Dye 工作溶液一起孵育 30 分钟。用 Hanks’ HEPES 缓冲液洗涤细胞两次后，向孔中加入含有 10 μmol/l CCCP 的培养基。孵育 24 小时后，通过荧光显微镜观察线粒体自噬。去除上清液后，向细胞中加入 250 μl 1 μmol/l Lyso Dye 工作溶液，并在 37oC 下孵育 30 分钟。用 Hanks 的 HEPES 缓冲液洗涤细胞两次，然后通过共聚焦荧光显微镜观察 Mtphagy、Lyso Dye 和 MitoBright Deep Red 的共定位。

使用 Parkin 表达的 HeLa 细胞（上图）和正常 HeLa 细胞（下）A、E）Mtphagy Dye 的荧光图像观察线粒体自噬； B, F) Lyso Dye 的荧光图像；C, G) MitoBright Deep Red 的荧光图像； D、H) 合并图像

线粒体膜电位变化的线粒体自噬诱导和检测
使用 Mitophagy 检测试剂盒 (MD01, MT02) 和 JC-1 MitoMP 检测试剂盒 (MT09) 将羰基氰化物间氯苯肼 (CCCP) 处理的表达 Parkin 的 HeLa 细胞中的线粒体状况与未处理的细胞进行比较。

结果：未经处理的细胞未检测到线粒体自噬，膜电位正常。然而，在处理的细胞中观察到膜电位和线粒体自噬的降低。

Experimental ConditionTransfection of Parkin plasmid to HeLa cells– HileyMax (H357) was used to transfect Parkin plasmid to HeLa cells (Parkin plasmid/HilyMax reagent: 0.1μg/0.2 μL) by incubating overnight.Detection of Mitophagy1. Add 0.1 μmol/L Mtphagy working solution to Parkin expressing HeLa cells and incubated for 30 minutes at 37 ℃2. Wash cells with HBSS3. Add 10 μg/mL CCCP/MEM solution and inclubate for 2 hours at 37 ℃4. Observe under fluorescence microscopeDetection of Mitochondrial Membrane Potential1. Add 10 μg/mL CCCP/MEM solution to Parkin expressing HeLa cells and incubate for 1.5 hours at 37 ℃2. Add 4 μmol/L JC-1 working solution (final concentration: 2 μmol/L) and incubate for 30 minutes at 37 ℃3. Wash with HBSS and add Imaging Buffer Solution.4. Observe under fluorescence microscope

Detecting Condition:

Mitophagy DetectionEx: 561 nm, Em: 570-700 nm

Mitochondrial Membrane Potential DetectionGreen Ex: 488 nm, Em: 500-550 nmRed Ex: 561 nm, Em: 560-610 nm

FCM

Mitochondrial contribution to lipofuscin formation

Procedure (Adherent cell):

1.Cells were washed twice with DMEM and afterwards incubated at 37 °C for 30 min with 100 nmol Mtphagy Dye diluted in DMEM.

2.After this incubation cells were again washed twice with DMEM followed by the addition of complete DMEM.

3.The induction of mitophagy was then accomplished by the addition of 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 24 h. Subsequently, fibroblasts were trypsinized and fluorescence intensity of Mtphagy Dye was measured by flow cytometry at 488 nm excitation and 655–730 nm emission.

Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy

Procedure (Suspension cell):

  1. Briefly, 2×106 CD4+ T cells were divided into four tubes containing serum-free RPMI.
  2. Tube #1 was for unstained cells that followed all washing and media changing processes.
  3. 100 nmol/l Mtphagy Dye working solution was added to tubes #2, 3, 4 and then all tubes were incubated at 37°C for 30 minutes.
  4. The cells were then washed with serum-free medium. After discarding the supernatant, complete medium (RPMI 1640, 10% FBS, 1% P/S/G) was added to all the tubes.
  5. Ten μmol/l CCCP (Sigma-Aldrich) mitophagy-inducer was then added to tube #3, 100 nmol/l Bafilomycin A1 (Sigma-Aldrich) autophagy inhibitor was added to tube #4.
  6. All tubes were then incubated at 37 °C for 18 hours.
  7. After 18 hours, cells were washed with FACS buffer.
  8. Mtphagy Dye fluorescence detection by flow cytometry (BD FACSCANTO II) was performed using 488 nm for excitation and 695 nm for emission (this corresponds to the PerCP cy5.5 channel). Data were analyzed using FLOWJO software (version 10).

Microplate Reader

PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: involvement of ROS and mitophagy

No. Sample Instruments Publications
1) Cell(HeLa, MEF) Fluorescencemicroscope H. Iwashita, H. T. Sakurai, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, K. Okuma, S. Shimizu, and Y. Ueno, “Small fluorescent molecules for monitoring autophagic flux.”, FEBS Letters., 2018, 592, (4), 559–567.
2) Cell(HeLa) Fluorescencemicroscope T. Sakata, A. Saito and H. Sugimoto, “In situ measurement of autophagy under nutrient starvation based on interfacial pH sensing.”, Scientific Reports., 2018, 8, 8282.
3) Cell(HS-MM) Fluorescencemicroscope Y. Egawa, C. Saigo, Y. Kito, T. Moriki and T. Takeuchi , “Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma.”, PLoS One., 2018, 13, (6), e0198940.
4) Cell(HaCaT) Fluorescencemicroscope S. Abe, S. Hirose, M. Nishitani, I. Yoshida, M. Tsukayama, A. Tsuji and K. Yuasa , “Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells.“, Biosci. Biotechnol. Biochem. ., 2018, 82, (12), 1347.
5) Cell(KGN) Fluorescencemicroscope W. Yuping, M. Congshun, Z. Huihui, Z. Yuxia, C. Zhenguo and W. Liping, “Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage.”, Reprod. Biol. Endocrinol., 2018,doi: 10.1186/s12958-018-0404-4.
6) Cell(BmN) Fluorescencemicroscope S. Xue, F. Mao, D. Hu, H. Yan, J. Lei, E. Obeng, Y. Zhou, Y. Quan, and W. Yu, “Acetylation of BmAtg8 inhibits starvation-induced autophagy initiation.”, Mol. Cell Biochem., 2019,doi: 10.1007/s11010-019-03513-y.
7) Cell(HeLa) Fluorescence microscope F. Hongbao,Y. Shankun, C. Qixin, L. Chunyan, C. Yuqi, G. Shanshan, B. Yang, T. Zhiqi, L. Z. Amanda, T. Takanori, C.Yuncong, G. Zijian, H. Weijiang and D. Jiajie , “De Novo-Designed Near-Infrared Nanoaggregates for Super-Resolution Monitoring of Lysosomes in Cells, in Whole Organoids, and in Vivo.”, ACS Nano, 2019, 13, (12), 1446.
8) Cell(RT-7) FlowCytometer E. Sasabe, A. Tomomura, N. Kitamura and T. Yamamoto, “Metal nanoparticles-induced activation of NLRP3 inflammasome in human oral keratinocytes is a possible mechanism of oral lichenoid lesions.”, Toxicol In Vitro., 2020, 62, 104663.
9) Cell(multiple myeloma) Fluorescence microscope J. Xia, Y. He, B. Meng, S. Chen, J. Zhang, X. Wu, Y. Zhu, Y. Shen, X. Feng, Y. Guan, C. Kuang, J. Guo, Q. Lei, Y. Wu, G. An, G. Li, L. Qiu, F. Zhan and W. Zhou, “NEK2 induces autophagy-mediated bortezomib resistance by stabilizing Beclin-1 in multiple myeloma.”, Mol Oncol, 2020, DOI: 10.1002/1878-0261.12641.
10) Cell(Human L2) Fluorescence microscope Q. Xu, W. Shi, P. Lv, W. Meng, G. Mao, C. Gong, Y. Chen, Y. Wei, X. He, J. Zhao, H. Han, M. Sun and K. Xiao, “Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy.”, Cell Death Dis., 2020, 11(1), 6.
11) Cell(Cardiomyocytes) Fluorescence microscope L Cui, LP Zhao, JY Ye, L Yang, Y Huang, X.P. Jiang, Q. Zhang, JZ. Jia, DX. Zhang and Y. Huang, “The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes.“, Front Cell Dev Biol, 2020,DOI:10.3389/fcell.2020.00031.
12) Cell(IPEC-J2) Fluorescence microscope Y Yang, J Huang, J Li, H Yang and Y. Yin, “The Effects of Butyric Acid on the Differentiation, Proliferation, Apoptosis, and Autophagy of IPEC-J2 Cells..”, Curr. Mol. Med., 2020, 20(4), 307.
13) Cell(Fibroblasts, Kidney epithelial cells) Fluorescence microscope M. M. Ivanova, J. Dao, N. Kasaci, B. Adewale, J. Fikry and O. G. Alpan  , “Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy”, Biomolecules , 2020,  10(6), 837.
14) Cell(NHEKs) Fluorescence microscope S. Ikeoka and A. Kiso  , “The Involvement of Mitophagy in the Prevention of UV-B-Induced Damage in Human Epidermal Keratinocytes “, J. Soc. Cosmet. Chem. Jpn., 2020,  54(3), 252.

 

与 Keima-Red 相比，该套件有什么优势？
我们的试剂盒使用小分子荧光探针，可以在不表达荧光蛋白的情况下检测线粒体自噬现象。我可以将 Mitophagy 检测试剂盒与固定细胞一起使用吗？
不，该试剂盒适用于活细胞，因为 Mtphagy Dye 会在完整的线粒体中积累。为什么不能将 Mtphagy 染料和 Lyso 染料混合在一起？
由于 Lyso 染料的荧光在长时间染色后会消失，如果将两种染料一起添加，Lyso 染料不会发出足够的荧光来进行双重染色。因此，在观察之前添加溶血染料。

我可以用 Mitophagy 检测试剂盒检测多少次？
对于 96 孔板格式（100 ul/孔），该试剂盒足以容纳 5 个板。对于 35 mm 培养皿格式 (2ml)，该试剂盒足以进行 25 次检测。

成功检测的一些技术技巧是什么？
1. 在诱导线粒体自噬之前加入 Mtphagy Dye 工作溶液（Mtphagy Dye 在完整的线粒体中积累）2。使用适当的过滤器 – Mtphagy 染料：Ex.500-560 nm、Em.670-730 nm 用于显微镜检测3。阳性对照使用 CCCP 或 FCCP*FCCP 或 CCCP 浓度：10 uM-30 uM，37℃孵育时间 3 小时 FCCP：羰基氰 4-（三甲基甲氧基）苯腙CCCP：羰基氰 3-氯苯腙4。如果可用，请使用共聚焦显微镜

培养基中的血清对活细胞成像敏感吗？
是的，它很敏感，因为 Mtphagy Dye 和 Lyso Dye 都会干扰培养基中的血清。请使用 Hank 的 HEPES 缓冲液或无血清培养基。

酚红有什么作用？
图 1. 荧光显微镜观察到的酚红引起的背景。但是，使用共聚焦显微镜时，酚红没有影响。我们强烈建议在检测线粒体自噬时使用共聚焦显微镜。

推荐的过滤器是什么？
Mtphagy Dye: Ex.500~560 nm, Em.670~730 nm, Lyso Dye: Ex.350~450 nm, Em.500~560 nm

使用 MitoBright Deep Red 进行多重染色有什么需要注意的地方吗？
Mtphagy Dye 的发射与 MitoBright Deep Red 的发射重叠。您需要在不激发 MitoBright Deep Red 的波长处激发 Mtphagy Dye。[示例] 染色 MitoBright Deep Red 后，在以下 Mtpahgy 染料和 MitoBright Deep Red 的激发和发射滤光片上观察细胞。

在 Mtphagy Dye 通道中检测到来自 MitoBright Deep Red 的荧光。请参阅以下部分以将光谱重叠减少到可接受的水平。1。更换激发滤光片

请将 Mtphagy Dye 的激发波长更改为大约 500 nm，并确保 MitoBright Deep Red 不被激发。 2.调整荧光检测的激发强度和灵敏度

请调整激发强度或检测灵敏度，直到不再检测到来自其他染料的荧光。之后，请仔细检查您是否可以检测到 Mtphagy Dye 的荧光。

如何检查光谱重叠使用 Mtphagy Dye、Lyso Dye 和 MitoBright Deep Red1 的示例。在 3 个不同的培养皿或孔中准备细胞（培养皿/孔 1-3 号）。2。在 1 号培养皿/孔中加入 Mtphagy Dye，在无血清培养基的 2 号培养皿/孔中加入 MitoBright Deep Red。 3.将细胞在 37°C 下孵育 30 分钟。 4.在 mitophagy 诱导条件下培养细胞（例如饥饿诱导 mitophagy）5。用无血清培养基将 Lyso Dye 添加到 3 号（3 号是培养皿/孔中仅包含在细胞中）。 6。将细胞在 37°C 下孵育 30 分钟。 7.在适当的 Ex./Em 处检测荧光。每种试剂的波长。Mtphagy 染料：例如。 500-560nm，时间。 670-730nmLyso 染料：ex. 350-450nm，时间。 500-560nmMitoBright 深红：ex. 640纳米，时间。 656-700nm8。检查是否在其他试剂的检测条件下观察到荧光。（例如：对于 Dish/well No.1，请检查在 Lyso 染料和 MitoBright Deep Red 的检测条件下观察到荧光。）

Dojindo,生物素标记试剂盒-NH2/1/LK03,Biotin Labeling Kit

Biotin Labeling Kit-NH2主要用于制备生物素标记的IgG，用于酶免疫分析（EIA）。 NH2 反应性生物素是该试剂盒的一个组成部分，具有与蛋白质或其他分子上的氨基反应的琥珀酰亚胺基 (NHS)（图 1）。 该试剂盒包含标记所需的所有试剂。 标记过程非常简单。 只需将 NH2 反应性生物素添加到 IgG 溶液中并在 37oC 下孵育 10 分钟。 每个 IgG 分子平均有 5 到 8 个生物素分子结合。 每个蛋白质的生物素分子数可以通过 HABA 测定法确定。 多余的生物素分子可以通过过滤管去除。

Precaution:♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.♦ IgG or biotin-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with molecular weights larger than 10,000, such as BSA or gelatin, purify the IgG solution before labeling biotin with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

Easily Switch Fluorescence wavelength on your primary antibody

1. Y. Kubota, Y. Oike, S. Satoh, Y. Tabata, Y. Niikura, T. Morisada, M. Akao, T. Urano, Y. Ito, T. Miyamoto, N. Nagai, G. Y. Koh, S. Watanabe and T. Suda, “Cooperative Interaction of Angiopoietin-like Proteins 1 and 2 in Zebrafish Vascular Development”, Proc. Natl. Acad. Sci. USA, 2005, 102, 13502.2. T. Yamabuki, A. Takano, S. Hayama, N. Ishikawa, T. Kato, M. Miyamoto, T. Ito, H. Ito, Y. Miyagi, H. Nakayama, M. Fujita, M. Hosokawa, E. Tsuchiya, N. Kohno, S. Kondo, Y. Nakamura, and Y. Daigo, “Dikkopf-1 as a Novel Serologic and Prognostic Biomarker for Lung and Esophageal Carcinomas”, Cancer Res., 2007, 67, 2517.3. H. Kohara, Y. Omatsu, T. Suhiyama, M. Noda, N. Fujii and T. Nagasawa, “Development of Plasmacytoid Dendritic Cells in Bone Marrow Stromal Cell niches Requires CXCL12-CXCR4 Chemokine Signaling”, Blood, 2007, 110, 4153.4. N. Ishikawa, A. Takano, W. Yasui, K. Inai, H. Nishimura, H. Ito, Y. Miyagi, H. Nakayama, M. Fujita, M. Hosokawa, E. Tsuchiya, N. Kohno, Y. Nakamura and Y. Daigo, “Cancer-testis Antigen Lymphocyte Antigen 6 Complex Locus K is a Serologic Biomarker and a Therapeutic Target for Lung and Esophageal Carcinomas”, Cancer Res., 2007, 67, 11601.5. A. Fukuda, T. Goto, K.N. Kuroishi, K.K. Gunjigake, S. Kataoka, S. Kobayashi and K. Yamaguchi, “Hemokinin-1 competitively inhibits substance P-induced stimulation of osteoclast formation and function”, Neuropeptides., 2013, 47, (4), 251.6. A. Ogawa , M. Sakatsume, X. Wang, Y. Sakamaki, Y. Tsubata, B. Alchi, T. Kuroda, H. Kawachi, I. Narita, F. Shimizu and F. Gejyo, “SM22alpha: the novel phenotype marker of injured glomerular epithelial cells in anti-glomerular basement membrane nephritis”, Nephron Exp. Nephrol.., 2007, 106, (3), e77.7. D. Men, T.T. Zhang, L.W. Hou, J. Zhou, Z.P. Zhang, Y.Y. Shi, J.L. Zhang, Z.Q. Cui, J.Y. Deng, D.B. Wang and X.E. Zhang, “Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay”, ACS Nano., 2015, 9, (11), 10852.8. D. Sugahara, Y. Kobayashi, Y. Akimoto, H. Kawakami, “Mouse intestinal niche cells express a distinct α1,2-fucosylated glycan recognized by a lectin from Burkholderia cenocepacia”, Glycobiology., 2017, 27, (3), 246.9. H. Sasaki-Iwaoka, M. Ohori, A. Imasato, K. Taguchi, K. Minoura, T. Saito, K. Kushima, E. Imamura, S. Kubo, S. Furukawa and T. Morokata, “Generation and characterization of a potent fully human monoclonal antibody against the interleukin-23 receptor”, Eur. J. Pharmacol.., 2018, 828, 89.10. K. Kaneshiro, M. Watanabe, K. Terasawa, H. Uchimura, Y. Fukuyama, S. Iwamoto, T.A. Sato, K. Shimizu, G. Tsujimoto and K. Tanaka, “Rapid quantitative profiling of N-glycan by the glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid”, Anal. Chem.., 2012, 84, (16), 7146.11. K.S. Tan, K. Kulkeaw, Y. Nakanishi, D. Sugiyama, “Expression of cytokine and extracellular matrix mRNAs in fetal hepatic stellate cells”, Genes Cells., 2017, 22, (9), 836.12. M. Tahara, S. Ohno, K. Sakai, Y. Ito, H. Fukuhara, K. Komase, M.A. Brindley, P.A. Rota, R.K. Plemper, K. Maenaka and M. Takeda, “The receptor-binding site of the measles virus hemagglutinin protein itself constitutes a conserved neutralizing epitope”, J. Virol.., 2013, 87, (6), 3583.13. M. Takahashi, Y. Ishida, D. Iwaki, K. Kanno, T. Suzuki, Y. Endo, Y. Homma and T. Fujita, “Essential role of Mannose-binding lectin-associated serine protease-1 in activation of the complement factor D”, J. Exp. Med.., 2010, 207, (1), 29.14. N. Hosen, Y. Matsunaga, K. Hasegawa, H. Matsuno, Y. Nakamura, M. Makita, K. Watanabe, M. Yoshida, K. Satoh, S. Morimoto, F. Fujiki, H. Nakajima, J. Nakata, S. Nishida, A. Tsuboi, Y. Oka, M. Manabe, H. Ichihara, Y. Aoyama, A. Mugitani , T. Nakao, M. Hino, R. Uchibori, K. Ozawa, Y. Baba, S. Terakura, N. Wada, E. Morii, J. Nishimura, K. Takeda, Y. Oji, H. Sugiyama, J. Takagi and A. Kumanogoh, “The activated conformation of integrin β7 is a novel multiple myeloma-specific target for CAR T cell therapy”, Nat. Med.., 2017, 23, (12), 1436.15. R. Nishino, A. Takano, H. Oshita, N. Ishikawa, H. Akiyama, H. Ito, H. Nakayama, Y. Miyagi, E. Tsuchiya, N. Kohno, Y. Nakamura and Y. Daigo, “Identification of Epstein-Barr virus–induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer”, Clin. Cancer Res.., 2011, 17, (19), 6272.16. T. Hiono, A. Matsuda, T. Wagatsuma, M. Okamatsu, Y. Sakoda and A. Kuno, “Lectin microarray analyses reveal host cell-specific glycan profiles of the hemagglutinins of influenza A viruses”, Virology., 2019, 527, 132.17. T. Oshima, S. Sato, J. Kato, Y. Ito, T. Watanabe, I. Tsuji, A. Hori, T. Kurokawa and T. Kokubo, “Nectin-2 is a potential target for antibody therapy of breast and ovarian cancers”, Mol. Cancer., 2013, 12, (60), .18. T. Yoshida, N. Shiraki, H. Baba, M. Goto, S. Fujiwara, K. Kume and S. Kume, “Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas”, Genes Cells., 2008, 13, (7), 667.19. X. Piao, T. Ozawa, H. Hamana, K. Shitaoka, A. Jin, H. Kishi and A. Muraguchi, “TRAIL-receptor 1 IgM antibodies strongly induce apoptosis in human cancer cells in vitro and in vivo”, Oncoimmunology., 2016, 5, (5), e1131380.20. Y. Shimazaki, Y. Kohno, “Successive analysis of antigen trapping and enzymatic digestion on membrane-immobilized avidin”, Anal. Biochem.., 2012, 422, (1), 55.

Determination of Biotin/ Protein RatioThe average number of biotin molecules per IgG molecule should be in the range of 5 and 8. If you need to determine the precise number of biotin molecules per Protein molecule use HABA assay. The following is a HABA assay protocol.

Reagent solution:200 μM HABA (4-hydroxyazobenzene-2-carboxylic acid) solution prepared with PBS, pH 7.4 …………………… 1 ml0.5 mg avidin/ml solution prepared with PBS, pH 7.4 ………………………………………………………………….. 1 mldiluted sample solution (55 μl biotinylated protein solution + 110 μl PBS, pH 7.4)25 μM biotin prepared with a mixed solution (2 volumes of PBS, pH 7.4 + 1 volume of WS buffer)……………… 200 μlPrepare solutions of various concentrations (12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM) with serial dilution …………. 200 μl/ea

Fig. 2 Typical Calibration Curve of HABA Assay

1. Mix HABA solution and avidin solution in a plastic tube.2. Add 100 μl of the HABA-avidin solution to 15 wells for multiple assays (n=3).3. Add 50 μl biotin solution (12.5 μM, 6.25 μM, 3.13 μM, and 1.56 μM) to 3 wells each and 50 μl of diluted sample solution to the rest of the 3 wells.4. Read the O.D. at 405 nm with a reference at 492 nm and prepare a calibration curve using the O.D. of various concentrations of biotin solution. Read the O.D. at 280 nm to determine the protein concentration. (e.g. molar absorptivity of IgG at 280 nm: 216,000).5. Determine the concentration of biotin in the sample solution and calculate the number of biotin molecules per protein.

我可以将此试剂盒用于其他蛋白质吗？
是的，如果分子量大于 50,000。在标记蛋白质之前是否必须使用过滤管？
如果蛋白溶液不含带氨基的小分子，蛋白浓度为10 mg/ml或约70 μM，则无需使用过滤管。只需将 10 μl 样品溶液与 90 μl 反应缓冲液混合，然后将混合物添加到一瓶 NH2 反应性生物素中。反应后，将所有反应混合物转移到过滤管中，然后按照从步骤 6 开始的方案进行操作。

我必须使用 WS 缓冲液来储存生物素标记的蛋白质吗？
您不必使用 WS 缓冲区。您可以根据您的实验选择任何类型的缓冲液。

我的样品含有少量不溶性物质。我该怎么办？
旋转样品并使用上清液进行标记。

生物素标记的蛋白质能稳定多久？
如果您将生物素标记的蛋白质储存在 0-5ºC，它可以稳定保存 2 个月。如需更长的储存时间，加入 100% 体积的甘油，等分，并储存在 -20ºC（如果蛋白质可以冷冻）。但是，请注意稳定性取决于蛋白质本身。

该试剂盒可以标记的最少 IgG 量是多少？
最低量为 10 μg IgG；只需遵循协议。 10 μg 至 100 μg IgG 的标记比率保持不变。

Dojindo,4-PDS/1/P017,2-PDS 和 4-PDS 用于生物样品中硫醇基的光度测定

2-PDS 和 4-PDS 用于生物样品中硫醇基的光度测定。 这些化合物在 pH 值为 5 时与硫醇基团反应形成巯基吡啶（图 1 和 2）。 2-Mercaptopyridine 和 4-Mercaptopyridine 的最大波长分别为 343 nm 和 324 nm。

Fig 1. 4-PDS reaction with thiol compound

1. D. R. Grassetti, et al., Determination of Sulfhydryl Groups with 2, 2 E or 4, 4 EDithiodipyridine. Arch Biochem Biophys. 1967;119:41-49.2. R. E. Humphrey, et al., Spectrophotometric Determination of Sulfite with 4, 4 EDithiodipyridine and 5, 5 EDithiobis-(2-nitrobenzoic acid). Anal Chem. 1970;42:698-702.3. R. E. Humphrey, et al., Spectrophotometric Determination of Cyanide with Organic Disulphides. Talanta. 1971;18:491-497.4. T. Uete, et al., Spectrophotometric Micromethod for Measuring Cholinesterase Activity in Serum or Plasma. Clin Chem. 1972;18:454-458.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,NTA-SAM Formation Reagent/2/N475,金属离子引入 NTA 生物分子或 NTA 化合物

NTA 与 Ni 等重金属形成稳定的络合物，因此可用于将金属离子引入 NTA 生物分子或 NTA 化合物。 这些金属螯合物通常用于检测与金属离子相互作用的特定物质，例如带组氨酸标签的蛋白质。

什么是 SAM 形成试剂？SAM（自组装单层）是通过化学吸附在固体表面形成的高度组织化的分子薄层。 由于其高适用性，SAM可用于生物传感器。 然而，在使用生物材料样品时，通常会观察到非特异性结合。 在大多数情况下，SAM 试剂的浓度和组合并未针对在金属表面上制备有组织的层进行优化。 Dojindo 提供的 SAM Formation Reagents 已在一瓶中优化且易于使用。 优化后的试剂可以显着减少非特异性结合。

Comparison DataDojindo SAM Formation Reagent gives us lower background and higher S/N ratio.

Data by QCM

Experimental Conditions:His-tagged ProteinA : 10 ug/ml His-tagged ProteinA in bufferBuffer : 10 mM HEPES (pH 7.2) ,150 mM NaCl , 0.5%Tween-20IgG : 5.5 ug/ml Rabbit IgG in buffer

Instrument： Affinix QNμ(QCM2008-LVKIT), INITIUM, Inc.

Leo JC, Oberhettinger P, Yoshimoto S, Udatha DB, Morth JP, Schutz M, Hori K, Linke D., “Secretion of the Intimin Passenger Domain is driven by Protein Folding.”, Journal of Biological Chemistry ., 2016, doi: 10.1074/jbc.M116.731497

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,BMPO/50/B568,BMPO 是作为一种自旋捕集试剂开发

自旋捕获分析是检测和识别短寿命自由基的最可靠技术之一。 EPR (ESR) 自旋阱试剂可检测体外和体内系统产生的超氧化物和羟基自由基。 BMPO 是作为一种自旋捕集试剂开发的，它可以加合超氧化物并显示出比其他自旋捕集剂更长的半衰期（t1/2=23 分钟）。它为我们提供了可重复且稳定的结果。由于 BMPO 易溶于水，亲水性样品适用于自由基分析。
通用协议测量芬顿反应中的羟基自由基1。用 5 ml ddH2O.2 溶解 1.5 mg BMPO。将 15 μl BMPO 溶液、75 μl 1 mM H2O2 和 75 μl 100 μM FeSO4 添加到 50 μl ddH2O.3 中。将溶液转移到 ESR 样品管中，并在一定时间后测量 ESR 光谱，例如1分钟.4。从峰高计算相对强度。测量黄嘌呤氧化酶 (XO) 反应中的超氧自由基1。用 1 ml 50 mM 磷酸盐缓冲液 (pH7.4).2 溶解 1 mg BMPO。制备含有 1 mM DTPA 和 0.4 mM Xanthine.3 的 50 mM 磷酸盐缓冲液 (pH 7.4)。制备含有 0.1 U/ml 黄嘌呤氧化酶 4 的 50 mM 磷酸盐缓冲液（pH 7.4）。混合 15 μl 溶液 A、135 μl 溶液 B 和 10 μl 溶液 C.5。将溶液转移到 ESR 样品管中，并在一定时间后测量 ESR 光谱，例如8 分钟。 6。从峰高计算相对强度。

Experimental Data from Bruker  >> Download the detail (PDF)Superoxide Detection

协议 1。 制备含有 25 μM 二乙烯三胺五乙酸 (DTPA) 作为过渡金属螯合剂的 100 mM 磷酸盐缓冲液 (pH 7.4) 溶液。2。 在 100 mM 磷酸盐缓冲液中配制 1 mM 次黄嘌呤溶液，pH 7.4.3。 配制浓度为 1 单位/ml4 的黄嘌呤氧化酶溶液。 将 10 mg BMPO 溶解到 200 µl 磷酸盐缓冲液中（最终浓度应为 250 mM），配制 BMPO 溶液。5。 将您的反应混合物制备成总反应体积为 200 µl.6。 在 Eppendorf tube.7 中加入 70 µl 缓冲液。 加入 20 µl 的 250 mM BMPO 溶液和 100 µl 的 1 mM 原液次黄嘌呤溶液。8。 用 10 µl 黄嘌呤氧化酶启动反应，涡旋管子并将溶液转移到平板细胞中。9。 将扁平电池插入腔中，调整光谱仪，并获取光谱。

组分的最终浓度为：25 mM BMPO、0.5 mM 次黄嘌呤和 0.05 单位/ml 黄嘌呤氧化酶。

Hydroxyl radical detection

 协议 1。配制 1 mM FeSO4、10 mM H2O2 和 250 mM BMPO 在水中的溶液。2。将您的反应混合物制备成总反应体积为 200 µl.3。向 Eppendorf 管中加入 140 µl 蒸馏水。4。添加 20 µl 250 mM BMPO 溶液和 20 µl FeSO4 原液 1 mM 溶液。5。用 20 µl 10 mM H2O2.6 启动反应。混合反应物并迅速将溶液转移到平板电池中。 7．将扁平电池插入腔中，调整光谱仪，并获取光谱。

组分的最终浓度为：25 mM BMPO、0.1 mM FeSO4 和 1 mM H2O2。给出了 BMPO 自旋捕获自由基的输出光谱。在 BMPO 的情况下，形成了 BMPO/OH 的两种立体异构体，类似于 BMPO/OOH 结果。两种 BMPO 加合物的构象 I 为 aN = 13.47 G、aHβ = 15.31 G 和 aHγ1 = 0.62 G，构象 II 为 aN = 13.56 G、aHβ = 12.3 G 和 aHγ1 = 0.66 G。

您应该始终执行排除一种或多种试剂的对照实验。这些实验将揭示任何顺磁性杂质，并将证明产生 EPR 信号所需的所有组件。

 

Time Course of the Superoxide Signal

Data and Protocol were kindly provided by Bruker Corporation.

After feeding the results of SpinFit into SpinCount, the concentration changes over the time of the experiment are obtained.

O2 的稳定性和反应数据。-DetectionBMPO 给出了不同实验条件下反应超氧化物的速率常数和相应自旋陷阱加合物的更长稳定性。链接到已发表论文

1. H. Zhao、J. Joseph、H. Zhang、H. Karoui 和 B. Kalyanaraman，固体环状硝酮自旋阱的合成和生化应用：用于检测超氧阴离子和谷胱甘肽的相对优越的阱。 自由基生物医学。 2001；31:599-606.2。 通用汽车 Rosen, P. Tsai, J. Weaver, S. Porasuphatana, L. Roman, A. A. Starkov, G. Fiskum 和 S. Pou，四氢生物蝶呤在调节神经元一氧化氮合酶产生的超氧化物中的作用。 J生物化学。 2002；277:40275-40280。

半衰期数据在 O2.- 检测中观察到长半衰期。

BMPO is applicable for experiments which requires longer handlin time. (ex. monitoring of enzyme reactions)

Dojindo,DMPO/1/D048,Dojindo 的 DMPO 的质量得到了很好的控制

由于潜在的癌症风险及其促进年龄的作用，活体内的自由基已成为一个经常研究的课题。 DMPO 是研究自由基最常用的自旋捕获试剂。 它适用于捕获氧自由基，尤其是超氧化物，并适用于生产具有特征 EPR (ESR) 模式的加合物。 然而，大多数市售 DMPO 含有会导致高背景的杂质。 因此，DMPO 需要进一步纯化才能在 EPR 上运行实验。 Dojindo 的 DMPO 的质量得到了很好的控制，Dojindo 的 DMPO 不需要任何预纯化过程。 没有杂质会导致背景问题。

通用协议评估超氧化物清除活动1。 将 15 μl DMPO 和 50 μl 5 mM 次黄嘌呤加入 35 μl 0.1 M 磷酸盐缓冲液 (pH 7.8).2。 加入 50 μl SOD 标准品或待测样品，涡旋 1-2 秒。 3． 立即加入 50 μl 的 0.4 U/ml 黄嘌呤氧化酶和 voltex.4。 将溶液转移到 ESR 样品管中，并在一定时间后测量 ESR 光谱，例如 1分钟.5。 从峰高计算相对强度（DMPO-O2-/Mn2+）。

C-, N-, and S-centered radicals Detection >> Download the detail (PDF)

DMPO is capable of trapping not only O-centered radicals but also C-, N-, and S-centered ones. The following experiments are used to demonstrate how different types of radicals can be trapped by DMPO in a simple HRP/H2O2 system by using different protein substrates.

Protocol 1. Prepare a solution of 100 mM phosphate buffer (pH 7.4) containing 25 µM diethylenetriaminepentaacetic acid (DTPA).2. Make up a solution of the following peroxidase substrates: (A) 100 mM sodium formate (HCOONa); (B) 100 mM potassium cyanide (KCN); (C) 100 mM sodium azide (NaN3); (D) 100 mM sodium sulfite (Na2SO3) in 100 mM phosphate buffer, pH 7.4.3. Make up a solution of horseradish peroxidase with concentration of 4.0 mg/ml (~ 100 µM) and 1 mM solution of hydrogen peroxide (H2O2).4. Make up a solution of DMPO with concentration of 1 M.5. Prepare your reaction mixture to a total reaction volume of 200 µl. Add 130 µl of buffer to an Eppendorf tube.6. Add 20 µl DMPO of your 1 M DMPO solution, 20 µl of one of the substrates Estock solutions, 10 µl of 1 mM H2O2, and initiate the reaction with 20 µl HRP.7. Vortex the tube, transfer the solution to a flat cell, and acquire the spectrum.8. The final concentrations of the components are: 100 mM DMPO, 10 mM substrate (formate, cyanide, azide, sulfite), 50 µM H2O2, and 10 µM HRP.Data and Protocol were kindly provided by Bruker Corporation.

Superoxide Detection Procedure

Protocol1. Prepare a solution of 100 mM phosphate buffer (pH 7.4) containing 25 µM diethylenetriaminepentaacetic acid (DTPA) as transition metal chelator. 2. Make up a solution of 1 mM hypoxanthine in 100 mM phosphate buffer, pH 7.4. 3. Make up a solution of xanthine oxidase with concentration of 1 unit/ml 4. Make up a solution of DMPO with concentration of 1 M. 5. Prepare your reaction mixture to a total reaction volume of 200 µl. 6. Add 70 µl of buffer to an Eppendorf tube. Add 20 µl DMPO of your 1 M DMPO solution and 100 µl hypoxanthine of the stock 1 mM solution. 7. Initiate the reaction with 10 µl xanthine oxidase, vortex the tube and transfer the solution to a flat cell. 8. Insert the flat cell into the cavity, tune the spectrometer, and acquire the spectrum.

The final concentrations of the components are: 100 mM DMPO, 0.5 mM hypoxanthine, and 0.05 units/ml xanthine oxidase.You should always perform control experiments in which one or more of the reagents are excluded. These experiments will reveal any paramagnetic impurities and will demonstrate that all the components were required to produce the EPR signal.Data and Protocol were kindly provided by Bruker Corporation.

1. S. Sankarapandi, et al., Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide. J Biol Chem. 1999;274:34576-34583.2. H. Li, et al., A pyrroline derivative of mexiletine offers marked protection against ischemia/reperfusion-induced myocardial contractile dysfunction. J Pharmacol Exp Ther. 2000;295:563-571.3. H. P. Souza, et al., Quantitation of superoxide generation and substrate utilization by vascular NAD(P)H oxidase. Am J Physiol Heart Circ Physiol. 2002;282:H466-H474.4. S. Kaewpila, et al., Manganese superoxide dismutase modulates hypoxia-inducible factor-1 alpha induction via superoxide. Cancer Res. 2008;68:2781-2788.5. M. L. T. Teoh, et al., Overexpression of extracellular superoxide dismutase attenuates heparanase expression and inhibits breast carcinoma cell growth and invasion. Cancer Res. 2009;69:6355-6363.6. Y. Song, et al., Nonenzymatic displacement of chlorine and formation of free radicals upon the reaction of glutathione with PCB quinones. PNAS. 2009;106:9725-9730.

Purity Data of DMPO by HPLCNo impurities(*) were observed in Dojindo’s DMPO.

Fig. 2 Comparison of the Purity by HPLC analysis

S/N Ratio in EPR (ESR) StudyComparison of EPR spectra between Dojindo’s DMPO and other suppliers. Spectra were taken in the presence of hydroxy radicals generated by Fenton reaction(black) and blank(blue). Dojindo’s DMPO gives very clear signal and higher S/N rate than supplier E and S.

Fig. 3 The signal and background from EPR detection

DescriptionImmuno-spin Trapping method was developed for detecting DNA and Protein radicals in biological analysis. ROS (Reactive Oxygen Species) produces modification of the structure and function of biomolecules that relate on the cause of variety diseases. To understand the mechanism of oxidative reactions, it is very important for analyze which molecules are involved in the oxidation process.DMPO is the most popular spin-trapping reagent that traps radicals in protein and DNA samples. The DMPO-Protein or DMPO-DNA nitrone adducts are determined using a ELISA, Western Blotting, Mass spectorometry, Imaging, and so on. The most of commercialized DMPO contains impurities that cause high backgrounds. Thus, DMPO requires further purification steps before use it. The quality of Dojindo’s DMPO is well controlled and Dojindo’s DMPO doesn’t require any pre-purification process. There are no impurities to cause a background problem.

Principle of Immuno-spin Trapping Method

Anti-DMPO IgGs are available from Abcam

Example Protocol : Radical DNA Detection► Referred PublicationDetection and imaging of the free radical DNA in cells–site-specific radical formation induced by Fenton chemistry and its repair in cellular DNA as seen by electron spin resonance, immuno-spin trapping and confocal microscopy. Bhattacharjee S, Chatterjee S, Jiang J, Sinha BK, Mason RP., Nucleic Acids Res. 2012, 40, 5477-86

► Evaluation of radical DNA by ELISA1. Extract DNA from RAW cells and dilute DNA to 5 μg/ml in PBS.2. Add 25μl of DNA solution and 25μl of Reacti-Bind DNA coating solution in each well of the plate and incubatefor 2 Eh at 37°C.3. Wash the wells once with washing buffer (PBS containing 0.05% non-fat dry milk and 0.1%Tween-20).4. Block with blocking buffer (PBS containing 3% non-fat dry milk) for 2h at 37°C5. Detect DMPO-DNA radical adduct with anti-DMPO and HRP-conjugated secondary antibody.6. After three washes, add the Immobilon chemiluminescence substrate each well and measure the intensity of luminesscense.

► Another Aplication in this paper• Cell Imaging

Example Protocol : Radical Protein Detection► Referred PublicationSuperoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling. Chen CA, Lin CH, Druhan LJ, Wang TY, Chen YR, Zweier JL., J Biol Chem. 2011, 286, 29098

► Evaluation of radical protein by cell imaging1. Prepare of Bovine aortic endothelial cells (104 cells) in 35-mm dishes.2. Add 10 μM Menadione and 50 mM DMPO and incuvate cells.3. Wash the cells with PBS and fix them with 3.7% paraformaldehyde for 10 minutes.4. Permeabilize the cells with 0.25% Triton X-100 in TBST(Tris buffered saline with Tween) for 10 minutes5. Block the cells with 5% goat serum in TBST.6. Visualize DMPO-protein radical adduct with anti-DMPO IgG and fluorescein labeled secondary antibody.7. Analyze Protein radicals by fluorescent microscopy.

 

Dojindo,Amine Coupling Kit/2/A515,胺偶联试剂盒

胺和羧酸偶联是通过共价键结合到生物传感器表面来固定蛋白质或肽的最常用方法之一。 Dojindo 的胺偶联试剂盒包含所有试剂和缓冲液，用于羧酸活化、蛋白质固定和封闭所需的试剂和缓冲液。 试剂盒中包含的封闭溶液通过封端残留的活化酯，最大限度地减少非特异性蛋白质对表面的吸收。 每个套件足以进行大约 40 次固定。

Kit contents:Unite: 2 ml x 4Contents:WSC 4 tubesNHS 4 tubesActivation buffer 20 ml x 1Reaction buffer 10 ml x 1Blocking solution 20 ml x 1

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,羧酸SAM形成试剂/1/C488,Dojindo 的胺偶联试剂盒包含所有试剂和缓冲液

胺和羧酸偶联是通过共价键结合到生物传感器表面来固定蛋白质或肽的最常用方法之一。 Dojindo 的胺偶联试剂盒包含所有试剂和缓冲液，用于羧酸活化、蛋白质固定和封闭所需的试剂和缓冲液。 试剂盒中包含的封闭溶液通过封端残留的活化酯，最大限度地减少非特异性蛋白质对表面的吸收。 每个套件足以进行大约 40 次固定。

Dojindo,BCECF-AM/1/B262,BCECF 是使用细胞内 pH 探针

BCECF 是最广泛使用的细胞内 pH 探针。 Tsien 博士和其他人通过引入两种额外的羧酸盐来改进这种羧基荧光素，使其能够更好地被细胞保留。 BCECF 是高度水溶性的，因为它在中性 pH 下具有 4 至 5 个负电荷；加载后变得难以通过细胞膜。其 pKa 值为 6.97，高于羧基荧光素。 BCECF在激发光谱中在439 nm处有一个等吸收点，因此可用于比率测定，类似于Fura 2。比率测定通常使用505 nm和439 nm的波长，设置490 nm和450 nm滤光片在激发光源前。 530 nm 滤光片用于其荧光信号。请注意，激发光谱与吸收光谱略有不同。 BCECF-AM 是 BCECF 的乙酰氧基甲酯，可以轻松地将 BCECF 加载到细胞中。 BCECF-AM 与其他乙酰氧基甲酯一样，仅通过孵育才能在细胞中积累。 BCECF-AM 对水分非常敏感；应该小心处理。 DMSO 溶液的颜色随着 AM 形式的分解从淡黄色变为深橙色。因此，可以通过颜色变化来监测 AM 酯的水解。

General Protocol (for Human Neutrophil)*Reagents:– 1 mM BCECF-AM/DMSO solution (1 mg BCECF in 1.45 ml DMSO)– HEPES buffer saline (20 mM HEPES, 153 mM NaCl, 5 mM KCl, 5 mM glucose, pH 7.4)

Protocol:1. Suspend cells in HEPES buffer solution to prepare 4×107 cells per ml.2. Add 1 mM BCECF-AM/DMSO solution to the cell suspension to prepare 3 μM BCECF-AM (1/300 vol of cell suspension) as the final concentration.3. Incubate the cell suspension at 37ºC for 30 minutes.4. Wash the cells 3 times with HEPES buffer saline and then prepare 3×106 cells per ml of the cell suspension.5. Determine the fluorescence intensity using a fluorescence microscope or a confocal laser microscope coupled with an image analyzer.

* Cell staining conditions depend on cell type, so it is necessary to optimize the conditions for each experiment

1. R. A. Steinhardt, et al., Development of K+-conductance and Membrane Potentials in Unfertilized Sea Urchin Eggs After Exposure to NH4OH. Nature. 1973;241:400-401.2. T. J. Rink, et al., Cytoplasmic pH and Free Mg2+ in Lymphocytes. J Cell Biol. 1982;95:189-196.3. A. M. Paradiso, et al., Na+ -H+ Exchange in Gastric Glands as Measured with a Cytoplasmic-trapped, Fluorescent pH Indicator. PNAS. 1984;81:7436-7440.4. S. Grinstein, et al., Phorbol Ester-induces Changes of Cytoplasmic pH in Neutrophils: Role of Exocytosis in Na+ – H+ Exchange. Am J Physiol. 1985;248:C379-C386.5. G. B. Zavoico, et al., Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs. J Biol Chem. 1986;261:13160-13167.6. G. R. Bright, et al., Fluorescence Ratio Imaging Microscopy: Temporal and Spatial Measurements of Cytoplasmic pH. J Cell Biol. 1987;104:1019-1033.7. C. Aalkjaer, et al., Intracellular pH Regulation in Resting and Contracting Segments of Rat Mesenteric Resistance Vessels. J Physiol. 1988;402:391-410.8. K. Tsujimoto, et al., Intracellular pH of Halobacteria Can Be Determined by the Fluorescent Dye 2 E 7 Ebis(carboxyethyl)-5(6)-carboxyfluorescein. Biochem Biophys Res Commun. 1988;155:123-129.9. M. A. Kolber, et al., Measurament of Cytotoxicity by Target Cell Release and Retention of the Fluorescent Dye Bis-carboxyethylcarboxyfluorescein(BCECF). J Immunol Methods. 1988;108:255-264.10. H. Harada, et al., cAMP Activates Cl-/HCO3 – Exchange for Regulation of Intracellular pH in Renal Epithelial Cells. Biochim Biophys Acta. 1991;1092:404-407.11. C. C. Freudenrich, et al., Intracellular pH Modulates Cytosolic Free Magnesium in Cultured Chicken Heart Cells. Am J Physiol. 1992;262:C1024-C1030.12. K. Khodakhah, et al., Functional Heterogeneity of Calcium Release by Inositol Triphosphate in Single Purkinje Neurones, Cultured Cerebellar Astorocytes, and Peripheral Tissues. PNAS. 1993;90:4976-4980.13. G. Boyarsky, et al., Superiority of in vitro Over in vivo Calibrations of BCECF in Vascular Smooth Muscle Cells. FASEB J. 1996;10:1205-1212.14. S. A. Weston, et al., New Fluorescent Dyes for Lymphocyte Migration Studies Analysis by Flow Cytometry and Fluorescent Microscopy. J Immunol Methods. 1990;133:87-97.15. L. S. De Clerck, et al., Use of Fluorescent Dyes in the Determination of Adherence of Human Leucocytes to Endothelial Cells and the Effects of Fluorochromes on Cellular Function. J Immunol Methods. 1994;172:115-124.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,DNA Damage Quantification Kit-AP Site Counting-/20/DK02,DNA损伤定量试剂盒

DNA 的氧化损伤是其与活性氧 (ROS)，特别是羟基自由基相互作用的结果。通过芬顿反应由超氧阴离子和过氧化氢产生的羟基自由基在 DNA 中产生多种修饰。羟基自由基对脱氧核糖部分的氧化攻击会导致 DNA 中游离碱基的释放，从而产生具有各种糖修饰和简单脱碱基位点（AP 位点）的链断裂。事实上，AP 位点是 ROS 产生的主要损伤类型之一。醛反应探针（ARP；N Eaminooxymethylcarbonylhydrazin-D-biotin）与存在于 AP 位点的开环形式上的醛基特异性反应（图 1）。该反应可以检测导致醛基形成的 DNA 修饰。用过量的 ARP 试剂处理后，DNA 上的所有 AP 位点都用生物素残基标记。这些生物素标记的 AP 位点可以使用亲和素-生物素测定进行量化，然后用与亲和素结合的过氧化物酶或碱性磷酸酶进行比色检测。 DNA 损伤定量试剂盒包含检测每 1 x 105 个碱基对 1 到 40 个 AP 位点所需的所有解决方案。

AP site Detection Principle

Mechanism of ARP Tagging at an Abasic Site

Recent Publications

Samples from Treatments References
MEF cells Cisplatin, Oxaliplatin Novel Role of Base Excision Repair in Mediating Cisplatin CytotoxicityA. Kothandapani, et al., J Biol Chem, 286, 14564(2011)
SF767glioblastoma AP endonuclease repair inhibitor Novel Small-Molecule Inhibitor of Apurinic/Apyrimidinic Endonuclease 1 Blocks Proliferation and Reduces Viability of Glioblastoma CellsA. Bapat, et al., J Pharmacol Exp Ther, 334, 988(2010)
hippocampaltissue global cerebral ischemia Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemiaR. A. Stetler, et al., PNAS, 107, 3204(2010)
CHO cells dominant-negative form of AP endonuclease 1 expressing Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and AntimetabolitesD. R. McNeill, et al., Mol Cancer Res, 7, 897(2009)
C57BL/6J mouse Valsartan Temporary Pretreatment With the Angiotensin II Type 1 Receptor Blocker, Valsartan, Prevents Ischemic Brain Damage Through an Increase in Capillary DensityJ. Li, et al., Stroke, 39, 2029(2008)

How to Prepare a Calibration Curve1. Calculate the average O.D. of each ARP-DNA standard solution.2. Subtract the blank O.D. from the average O.D.a)3. Plot the O.D. corresponding to the number of AP sites of the standard solution. X-axis is the number of AP sites and Y-axis is the O.D.4. Determine the number of AP sites in the sample using this calibration curve.

a) The blank O.D. is about 0.04-0.06 and the O.D. of the 40 ARP DNA standard solution is about 0.8-1.0. The O.D. value depends on HRPStreptavidin activity.

Fig. 3 Typical calibration curve of DNA Damage Quantification Kit

1. T. Lindahl, et al., Rate of Depurination of Native Deoxyribonucleic Acid. Biochemistry. 1972;11:3610-3618.2. M. Liuzzi, et al., A New Approach to the Study of the Base-excision Repair Pathway Using Methoxyamine. J Biol Chem. 1985;260:5252-5258.3. A. Sancar, et al., DNA Repair Enzymes. Annu Rev Biochem. 1988;57:29-67.4. M. Weinfeld, et al., Response of Phage T4 Polynucleotide Kinase Toward Dinucleotides Containing Apurinic Sites: Design of a 32P-postlabeling Assay for Apurinic Sites in DNA. Biochemistry. 1990;29:1737-1743.5. B. X. Chen, et al., Properties of a Monoclonal Antibody for the Detection of Abasic Sites, a Common DNA Lesion. Mutat Res. 1992;273:253-261.6. J. A. Gralnick, et al., The YggX Protein of Salmonella enterica Is Involoved in Fe(II) Trafficking and Minimizes the DNA Damage Cause by Hydroxyl Radicals:Residue CYS-7 is Essential for YggX Function. J Biol Chem. 2003;278:20708-20715.7. J. W. Pippin, et al., DNA Damage is a Novel Response to Sublytic Complement C5b-9 Induced Injury in Podocytes. J Clin Invest. 2003;111:877-885.8. S. Watanabe, et al., Methylated DNA-binding Domain 1 and Methylpurine DNA Glycosylase Link Transcriptional Repression and DNA Repair in Chromatin. PNAS. 2003;100:12859-12864.9. M. Endres, et al., Folate Deficiency Increases Postischemic Brain Injury. Stroke. 2005;36:321-325.10. J. Li, et al., Angiotensin II-Induced Neural Differentiation via Angiotensin II Type 2 (AT2) Receptor-MMS2 Cascade Involving Interaction between AT2 Receptor-Interacting Protein and Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatase 1. Mol Endocrinol. 2007;21:499-511.11. D. R. McNeill, et al., A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents. Mol Cancer Res. 2007;5:61-70.12. C. A. Downs, et al., Cellular pathology and histopathology of hypo-salinity exposure on the coral Stylophora pistillata. Sci Total Environ. 2009;407:4838-4851.13. C. A. Downs, et al., Symbiophagy as a cellular mechanism for coral bleaching. Autophagy. 2009;5:211-216.

Can I use single-stranded DNA or RNA?

No, you cannot use this kit to determine the number of abasic sites in single-stranded DNA or RNA. The O.D. reading of single-stranded DNA will be nearly twice that of double-stranded DNA because of the binding efficiency on the microplate.

How should genomic DNA be stored?

Prepare a DNA pellet and store at -20°C or -80°C if the DNA cannot be labeled with ARP immediately after isolation. After ARP labeling, the sample can be stored at 4°C in TE Buffer for several months.

How should I prepare the DNA?

You can use general protocols or commercially available DNA isolation kits. Between 2 to 4 abasic sites per 1 x 105 base pairs will be created during the DNA isolation process. Therefore, use the same isolation method to prepare each DNA sample.

What should I do if the sample DNA concentration is less than 100 μg per ml?

You can either use a filtration tube to concentrate your sample DNA or ethanol precipitation to recover DNA as a pellet and then re-dissolve it to prepare a 100 μg per ml solution.

What should I do if the sample DNA is less than 1 μg?

Add the same volume of ARP Solution and follow the manual. The recovery of the ARP-labeled DNA may be lower than the usual reactions, so measure the ARP-labeled DNA solution. The average recovery rate of the 0.5 μg DNA and 0.25 μg DNA is 70% and 50%, respectively.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,DPPH抗氧化剂检测试剂盒/500/D678,基于 Shimamura 等人报告的微孔板方法

身体抗氧化能力的变化与各种疾病和健康问题的发展有关。 因此，这增加了对具有抗氧化活性的食物（抗氧化食物）的期望。 高知大学的 Shimamura 等人。 开发了一种使用 DPPH（2,2-二苯基-1-苦基肼）评估抗氧化活性的方法，测量设备之间的差异很小 1）。 本产品使用基于 Shimamura 等人报告的微孔板方法。 通过手动 DPPH 测量方法和生产质量稳定的试剂盒，我们能够减少数据的可变性和试剂制备的复杂性，这是迄今为止遇到的问题。1） T. Shimamura 等人，肛门。 科学, 2014, 30, 717 – 721

Simple ProcedureDPPH and Trolox are unstable in solution. Please prepare immediately before using.DPPH content needs to be checked by measuring absorbance.Reagents necessary for measurement are inlcuded and subdivided. Begin experiments immediately with simple preparation before measurement.(Please use sonication to dissolve the DPPH Reagent.)

Measuring methodAfter preparing the reagent, add the reagent and sample to the 96-well microplate and react for 30 minutes.

Comparison with a conventional methodWhen measuring antioxidant activity with DPPH, the pH and solvent concentration in the solution affect the measurement. This product uses protocols and analysis methods to minimize these effects.

Effect of pHThe assay buffer provided with the product allows measurement at a certain pH.

Effect of sample solventThe sample volume is set to 1/10 (20 μL) of the total reaction solution. The kit is optimized so that there is no difference in the measured value even if the sample is dissolved in water or ethanol.

IC50 Value VariationIf the antioxidant capacity of a sample is analyzed using only IC50 values, the data will vary slightly in the different measurement conditions. By measuring the sample and standard substance (Trolox) simultaneously and calculating the antioxidant capacity as the Trolox equivalent activity value (TEAC), highly reproducible measurement values can be obtained.TEAC (μg TE/μg) = Trolox IC50 (μg/mL)/ Sample IC50 (μg/mL)”

Measurement ExampleConfirming differences between facilitiesAt three facilities, antioxidants were measured by the DPPH method.Gallic acid, catechin, and morin (known antioxidants) were measured with a spectrophotometer using a cuvette and calculated as a Trolox equivalent activity value (TEAC).There was nearly no difference in measured values between three facilities.Reference: T. Shimamura et al., NipponShokuhin Kagaku Kogaku Kaishi, 2007, 54, 482 – 487.

Comparison between microplate and cuvetteSimilar to the above experiment, three antioxidants were measured using a microplate and calculated as Trolox equivalent activity values. As a result, almost the same measurement results were obtained. This kit uses a microplate for measurement.

IC50 values vary, are there any precautions?
(1) Please make sure that all of the DPPH is dissolved.Please use sonication to dissolve the DPPH Reagent because it is difficult to dissolve.Transfer ethanol to the tube several times as described in the instruction manual. Confirm that the ethanol is no longer colored before use.(2) IC50 values may fluctuate greatly when the sample dilution interval is wide or when measured at the point where the inhibition curve is saturated (see below).Therefore, it is recommended that the optimal concentration range be confirmed in a preliminary experiment.
Can I dissolve DPPH by vortexing or pipetting?
Since DPPH is difficult to dissolve, it cannot be dissolved using vortex and pipetting.Be sure to use an ultrasonic cleaner because undissolved parts may cause variations in IC50.”
How many samples can I measure?

This depends on the kit size:

100 tests 500 tests
This kit contains enough reagents to measure one 96-well microplate (1 set of DPPH)Analyzing samples and standards in triplicate is recommended for accuracy.Therefore, the number of measurable samples is calculated as follows, assuming that measurement was performed at n: 3.DPPH Reagent per 1 kit is sufficient for 100 tests.Use for 1 sampleOne sample can be measured with the 100 test size kit.Number of wells used during preliminary experiment: Confirmation of optimum concentration range

Sample: 8 (dilution series of 8 points) X 3 (n: 3) = 24 wellBlank: 1 (Blank1) X 3 (n: 3) = 3 well

Number of wells used when calculating the IC50 value

Sample: 8 (dilution series of 8 points) X 3 (n: 3) = 24 well

Trolox: 4 (dilution series of 4 points) X 3 (n: 3) = 12 well

Blank: 1 (Blank1) X 3 (n: 3)= 3 well*If there is no require preliminary experiments, the additional sample measurement is possible.

 This kit contains enough reagents to measure 5 96-well microplates (5 sets of DPPH)Choose the quantity used based on your experiment (ex. 500 test x1 or 100 test x5)Up to 8 samples8 samples can be measured with the 500 test size kit.Number of wells during the preliminary experiment: Confirmation of optimum concentration rangeSample: 8 (dilution series of 8 points) X 3 (n: 3) X 8 (8 samples) = 192 wellBlank: 1 (Blank1) X 3 (n: 3) X 3 (3 plates) = 9 wellNumber of wells when calculating IC50 value

Sample: 8 (dilution series of 8 points) X 3 (n: 3) X 8 (8 samples) = 192 welll

Trolox: 4 (dilution series of 4 points) X 3 (n: 3) X 5 (5 plates) = 60 well

Blank: 1 (Blank1) X 3 (n: 3) X 3 (3 plates) = 9 well

IC50 value is known: 3 samples・If the IC50 value has already been measured with our kit, 3 samples can be measured with one kit.・If the IC50 value is not measured with our kit, it is recommended to perform a preliminary experiment as an unknown sample.

Number of wells when calculating the IC50 valueSample: 8（dilution series of 8 points) X 3 (n: 3) X 3 (Sample number) = 72 wellTrolox: 4 (dilution series of 4 points) X 3 (n: 3) = 12 wellBlank: 1 (Blank1) X 3 (n: 3) = 3 well

If the reaction time until measurement is increase, will the measured value be affected?
Longer reaction times can change the readings.In order to get the good results with reproducibility, measure immediately after incubation (25 ° C, 30 minutes, dark place) as described in the manual.When measuring multiple plates, please unify the measurement time for each plate.
What should I do if my sample is opaque?
If the sample is clear after the pretreatment described in the manual, measurement is possible.

Dojindo,11-Hydroxy-1-undecanethiol/10/H337

羟基烷硫醇用作金表面上的稀释试剂以控制反应基团的密度，或用作封闭剂以防止分析物在表面上的非特异性结合。新开发的 16-Hydroxy-1-hexadecanethiol 具有 16 个碳链，是市场上羟基烷硫醇中最长的烷硫醇。总共有 6 种不同的羟基烷硫醇，包括可用于金表面改性的羟基-EG6-十一烷硫醇和羟基-EG3-十一烷硫醇。当应用 16-Amino-1-hexadecanethiol 或 15-Carboxy-1-pentadecanethiol 时，16-Hydroxy-1-hexadecanethiol 用于制备均匀且高度定向的 SAM。 Herne 和他的同事在金表面上制造了硫醇衍生的单链 DNA (HS-ss-DNA) 和 6-Hydroxy-1-己硫醇的混合 SAM，以防止 HS-ss-DNA 的非特异性吸附。 Perez-Luna 和其他人在金表面上制作了生物素末端硫醇和 11-羟基-1-十一烷硫醇的混合 SAM。它们阻止了野生型链霉亲和素和链霉亲和素突变体的非特异性吸附。 Dubrovsky 及其同事使用 11-Hydroxy-1-十一硫醇控制了蛋白质在镀金硅胶表面上的非特异性吸附。他们提到了镀金硅胶在制备用于生物测定的明确的、表面功能化的支持物方面的有用性。

Dojindo,硫生物-蛋白质氧化还原状态监测试剂盒/5/SB11

蛋白质硫醇是细胞过程中的主要氧化还原信号。 氧化应激对蛋白质硫醇的修饰与细胞中的氧化还原状态有关。 该试剂盒的方法是准确检测硫醇残基的数量，以研究氧化应激环境中的氧化还原状态。

Mechanism of Thiol Modification with Protein-SHifter:

Fig. 2 Maleimide group of the Protein-SHifter binds to a free thiol group of a protein. The conjugate consists of a unique molecular weight that clearly visualized in electrophoresis.

Visualization of Redox State of Thiol Residues in a Protein:

Fig. 3 One molecule of Protein-SHifter binds to one molecule of free-thiol in protein. The number of the conjugation is directly proportional to the number of free thiol as clear band-shift.

Comparison Data:Linearity of mobility shifts derived from Protein SHifter comparing with PEG-Maleimide (PEG-Mal)

*Conjugation with Thioredoxin

Clear band-shift observed from Protein SHIfter comparing with PEG-Mal.

Detection of Redox State of Thiols residues in GAPDH

15% SDS-polyacrylamidegel

1. Satoshi Hara,Tatsuya Nojima,Kohji Seio, Masasuke Yoshida,Toru Hisabori.”DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specificprotein”Biochimical et Biophysical Acta,2013,1830(4) 3077.2. Satoshi Hara,Yuki Tatenaka, Yuya Ohuchi, Toru Hisabori,”Direct determination of the redox status of cysteine residues in proteins in vivo”. Biochimical and Biophysical Research Communications.2014,In Press.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,GSSG/GSH Quantification Kit/200/G257

谷胱甘肽（γ-L-glutamyl-L-cysteinylglycine）是一种存在于体内的三肽，作为谷胱甘肽过氧化物酶、谷胱甘肽S-转移酶、硫醇转移酶等的酶底物参与抗氧化、药物代谢等。谷胱甘肽通常以还原形式 (GSH) 存在，但 GSH 通过氧化应激等刺激转化为其氧化形式 (GSSG)。因此，GSH 和 GSSG 的比值已被记录为氧化应激的指标。GSSG/GSH 定量试剂盒包含 GSH 的掩蔽试剂。只需添加掩蔽试剂即可使样品中的 GSH 失活。因此，使用酶回收系统通过测量 DTNB (5,5 Edithiobis (2-nitrobenzoic acid) 的吸收 (λmax = 412 nm) 仅检测 GSSG。此外，GSH 可以通过从 GSSG 中减去来确定数量谷胱甘肽的总量。该试剂盒仅限于定量 0.5 μmol/l 至 50 μmol/l 的 GSH/GSSG 浓度和 0.5 μmol/l 至 25 μmol/l 的 GSSG 浓度。

Principle

Selective QuantificationAlthough conventional masking reagent, 2-Vinylpyridine(2-VP), interferes the reaction of GSSG measurement, Dojindo’s masking reagent does not interfere the reaction of GSSG measurement. Therefore, the exact ratio of GSSG and GSH is obtained with Dojindo GSSG/GSH detection Kit.

Measurement of GSSG with or without GSH masking reagent

InterferenceReducing agents such as ascorbic acid, β-mercaptoethanol, dithiothreitol (DTT) and cysteine, or thiol reactive compounds such as maleimide compounds, interfere with the glutathione assay. Therefore, SH compounds, reducing agents and SH reactive materials should be avoided during the sample preparation.

Required Equipment and Materials– Plate reader (405 or 415 nm filter)– 96-well microplate– Incubator (37ºC)– 20 µl and 200 µl pipettes, a multi channel pipette– 5-Sulfosalicylic Acid (SSA) Solution– Ethanol

Assay Procedure

Literature used “GSSG/GSH Quantification Kit”

Diurnal Variation of cadmium-induced mortality in miceN. Miura, Y. Yanagiba, K. Ohtani, M. Mita, M. Togawa, and T. Hasegawa, J. Toxicol. Sci., 2012, 37(1), 191<Detailed information on this article>– Measuring object: Hepatic GSH– Sample: Liver (Mice)– Preparation of sample1) Homogenize liver sample in 5% SSA for 30 seconds in ice-water bath.2) Centrifuge the homogenates consisting of 100 mg liver in 1 ml (10%) at 8,000 x g for 10 min. at 4°C to remove proteins.3) Assay with supernatants for GSH using GSSG/GSH Quantification Kit according to the manufacture’s instruction.

Effect of Oxidative Stress on Secretory Function in Salivary Gland CellsK. Okabayashi, T. Narita, Y. Takahashi, H. Sugiya, (2012), Oxidative Stress – Environmental Induction and Dietary Antioxidants, Edited by Volodymyr I. Lushchak, ISBN 978-953-51-0553-4, Hard cover, 388 pages, Publisher: InTech<Detailed information on this article>– Measuring object: GSH– Sample: Parotid acinar cells– Preparation of sample1) Collect parotid acinar cells by centrifugation at 10,000 g for 15 s and immediately mixed with 160 ul of 10 mM HCl.2) Freeze and thraw the mixture three times over, then mix with 40 ul of 5% SSA and then centrifuge at 8,000 g for 10 minutes.3) Collect the supernatant and dilute twice.

Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii Y. Kabeya, and S. Miyagishima, Plant Physiol., 2013, 161, 2102<Detailed information on this article>– Measuring object: GSH and GSSG– Sample: Chlamydomonas reinhardtii– Preparation of sample1) Collect Chlamydomonas reinhardtii cells by 1,000g for 5 min and wash with PBS.2) Resuspended in 5% SSA solution and disrupted by sonication.3) Collect the supernatants by 15,000g for 5 min.4) Isolate chloroplst by 700g for 5 min and wash with 50 mM HEPES-KOH, pH 7.5, containing 300mM sorbitol.5) Resuspend in 5% SSA solution and centrifuge at 15,000g for 5 min

Selected publicationsER Stress Cooperates with Hypernutrition toTrigger TNF-Depe. Cancer Cell. 2014;26:331-343.Purpose: Determine indicator of ER Stress in Liver

Mechanisms of cadmium-inducedchronotoxicity in mice. The journal of toxicological. 2013;38:947-957.Purpose: Determine the GSH/GSSG ratio in cadmium exposed Liver

Hepatitis C Virus Core Protein SuppressesMitophagy by Interacting with Parkin in the Context of MitochondrialDepolarization. The American Journal of Pathology. 2014;184:3026-3039.Purpose: Measure mitochondrial oxidative status in liver

Possible involvement of glutathione balance disruption in dihydropyrazine-induced cytotoxicity on human hepatoma HepG2 cells.The journal of toxicological.2012;37:1065-1069.Purpose: Investigate fluctuation of GSH/GSSG ratio in the cytotoxicity HepG2 Cells

Induction of heme oxygenase-1 contributes to survival of Mycobacterium abscessus in humanmacrophages-likeTHP-1cells.Redox Biology. 2015;4:328-339.Purpose: Determine redox state in macrophages

Age-related changes in salivary biomarkers.Journal of Dental Sciences. 2014;9:85-90.Purpose: Measure GSH/GSSG ratio in salivary

Protective effects of hydrogen sulfideanions against acetaminophen-induced hepatotoxicity in mice. The journal oftoxicological. 2015;40:837-841.Purpose: Measure glutathione of Hepatocyte

1. N. Kubota, et al., A high-fat diet and multiple administration of carbon tetrachloride induces liver injury and pathological features associated with non-alcoholic steatohepatitis in mice. Clin Exp Pharmacol Physiol. 2013; 40(7): 422-302. T. Tomofuji, et al., Supplementation of broccoli or Bifidobacterium longum-fermented broccoli suppresses serum lipid peroxidation and osteoclast differentiation on alveolar bone surface in rats fed a high-cholesterol diet. Nutr Res. 2012; 32(4): 301-73. T. Miyayama., et al., Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology. 2013; 8;305: 20-9.4. H. Miwa, et al., Leukemia cells demonstrate a differen t metabolic perturbation provoked by 2-deoxyglucose. Oncol Rep. 2013; 29(5): 2053-7.5. K. Unno, et al., Acute enhancement of non-rapid eye movement sleep in rats after drinking water contaminated with cadmium chloride. J Appl Toxicol. 2013 24.6. Y. Ishihara, et al., Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures. Neurochem Int. 2012; 60(8): 782-90.7. H. Miyamoto, et al., Thermophile-fermented compost as a possible scavenging feed additive to prevent peroxidation. J Biosci Bioeng. 2013; 116(2): 203-8.8. E. Taniai, et al., Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats. Toxicology Letters, October 2013.9.　G. Tian, et al., Ubiquinol-10 Supplementation Activates Mitochondria Functions to Decelerate Senescence in Senescence Accelerated Mice. Antioxidants & Redox Signaling, October 2013.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,ALPS/1/OC04

1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982;30:2492-2497.2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982;136:121-127.3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984;56:2849-2850.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,硫生物制剂-蛋白质氧化还原状态监测试剂盒Plus/20/SB12

蛋白质硫醇是细胞过程中的主要氧化还原信号。 氧化应激对蛋白质硫醇的修饰与细胞中的氧化还原状态有关。 该试剂盒的方法是准确检测硫醇残基的数量，以研究氧化应激环境中的氧化还原状态。

Fig. 1 Redox States of Thiol group(s) in Protein

Mechanism of Thiol Modification with Protein-SHifter:

Fig. 2 Maleimido group of the Protein-SHifter binds to a free thiol group of a protein. The conjugate consists of a unique molecular weight that shifts properly in electrophoresis. Photocleavage method enhances membrane transcription efficiency in western blotting.

Visualization of Redox State of Thiol Residues in a Protein:

Fig. 3 One molecule of Protein-SHifter binds to one molecule of free-thiol in protein. The number of the conjugation is directly proportional to the number of free thiol as clear band-shift.

Detection of the number of thiol on the GAPDH in HeLa cell lysate:

1. Satoshi Hara,Tatsuya Nojima,Kohji Seio, Masasuke Yoshida,Toru Hisabori.”DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specificprotein” Biochimical et Biophysical Acta ,2013,1830(4) 3077.2. Satoshi Hara,Yuki Tatenaka, Yuya Ohuchi, Toru Hisabori,”Direct determination of the redox status of cysteine residues in proteins in vivo”. Biochimical and Biophysical Research Communications. 2014,In Press.3. Yuichiro J. Suzuki, Faisal Almansour, Camilla Cucinotta, Vladyslava Rybka, Lucia Marcocci,”Cell signaling promoting protein carbonylation does not cause sulfhydryl oxidation: Implications to the mechanism of redox signaling”. F1000Research 2017, 6:4554. Yuichiro J. Suzuki, Lucia Marcocci, Takashi Shimomura, Yuki Tatenaka, Yuya Ohuchi, Tinatin I. Brelidze. “Protein Redox State Monitoring Studies of Thiol Reactivity”. Antioxidants. 2019 May ;8(5): 143.

Q. What is the adequate concentration of proteins and thiols in my sample?A. Adjust the protein concentration about 0.1 to 1 mg / mL and the thiol concentration to 100 μmol / mL or less.Please refer to the following protocol for a quantification example of thiol concentration (for 96 well plate) using DTNB (product code: D029).1. Reagent and solution preparationRequired material: Reaction buffer (100 mmol / L sodium phosphate, pH 8.0, 1 mmol / L EDTA)A) 400 μmol / L DTNB solution(1) Weigh 4 mg DTNB [5,5′-Dithiobis (2-nitrobenzoic acid), Code: D029] and dissolve in 1 mL of reaction buffer. (10 mmol / L) solution (a)(2) Dilute the solution (a) 25 times with reaction buffer. Final concentration: 400 μmol / L* Prepare 0.1 mL of solution for each well. (ex. if you use 9 wells, please prepare about 1mL.)B) L-Cysteine standard solution(1) Dissolve 2.42 mg of L-Cysteine in 5 mL of reaction buffer to make 4 mmol / L of L-Cysteine solution. Solution (b)(2) Dilute 100 μL of Solution (b) with 900 μL of reaction buffer to make a 400 μmol / L of L-Cysteine solution. Dilute this solution twice in order to make L-Cysteine standard solution (200, 100, 50, 25, 12.5, 0 μmol / L).C) Measurement samplePrepare several samples (cell lysate, protein solution, etc.) diluted with reaction buffer. (To measure at N = 3, prepare 350 μL or more.)2. Procedure(1) Add 100 μL of the measurement sample and L-Cysteine standard solution to each well of a 96-well microplate (Figure 1).(2) Add 100 μL of 400 μmol / L DTNB solution and mix by pipetting.(3) After incubating at room temperature for 15 minutes, measure the absorbance at 412 nm with a microplate reader.(4) Calculate the thiol concentration in the measurement sample from the L-Cysteine calibration curve (Figure 2).*Calculate the thiol concentration in the sample from the sample dilution ratio.

Dojindo,DAB/5/D006,DAB 被氧化成棕色色素

DAB 是免疫组化中检测过氧化物酶最常用的氧化显色染料之一。 在 H2O2 和过氧化物酶的存在下，DAB 被氧化成棕色色素。 这些棕色色素是苯胺黑类化合物，牢固地沉积在细胞膜或组织上的过氧化物酶周围。 由于加工或储存过程中的氧化，市场上的大多数 DAB 呈现棕色。 另一方面，Dojindo 提供高质量的 DAB，外观呈白色或略带红灰色的粉末，适合锐利染色。

Preparation of Sample Staining Solution1. Dissolve 9 mg DAB with 1 ml PBS to prepare 100X DAB solution.2. Mix 5 μl of 30% hydrogen peroxidase solution with 1 ml PBS to prepare 200X H2O2 solution.3. Add 10 ul of 100X DAB solution and 5 ul of 200X H2O2 solution to 1 ml PBS to prepare staining solutiona).4. Add the staining solution to a sample in a staining chamber, and incubate the sample at room temperature for 5 minutes to 1 hourb).5. Wash the sample with PBS several times to stop the staining reaction.

a)The staining solution is not stable. Prepare fresh solution prior to use.b)For better staining, incubate the sample with 0.09 mg DAB/PBS solution for 10 minutes before adding the staining solution.

1. A. B. Novikoff, et al., Studies on Microperoxisomes V. Are Microperoxisomes Ubiquitous in Mammalian Cells. J Histochem Cytochem. 1973;21:737.2. V. Herzog, et al., A New Sensitive Colorimetric Assay for Peroxidaase Using 3, 3 EDiaminobenzidine as Hydrogen Donor. Anal Biochem. 1973;55:554.3. H. Yamada, et al., Improvement of Technique of Immunohistochemical Demonstraction of Bioactive Substances in the Central Nervous System. Acta Histochem Cytochem. 1987;20:629.4. K. L. Cheng, Determination of Traces of Selenium 3,3 EDiaminobenzidine as Selenium(IV) Organic Reagent. Anal Chem. 1956;28:1738.

Cell Staining Redox Dyes

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,羧基-EG6-十六烷基硫醇/10/C463

聚乙二醇 (PEG) 广泛用于材料改性以提高表面的亲水性。 PEG 涂层材料通常在生理条件下更稳定。 由于 Carboxy-EG6-十一硫醇在末端有 6 个乙二醇单元、11 个碳原子和一个 SH 基团，因此它可用于在金表面上制备高度定向和亲水性的 SAM，适用于表面上的生物材料标记 改善的亲水性。 亲水表面可以防止蛋白质或其他生物材料发生非特异性结合。 因此，该试剂制备的SAM将为开发生物材料传感器或DNA/蛋白质微阵列提供更好的表面。 为了在金表面上制备羧基-EG6-SAM，使用羟基-EGn-十一硫醇（n=3, 6）根据引入到表面上的分子的密度来稀释羧基的数量。 有几篇关于标记蛋白质（如卵清蛋白和细胞色素 C）的论文。

1. C. Pale-Grosdemange, E. S. Simon, K. L. Prime, and G. M. Whitesides, Anal. Chem., 1999, 71, 777-790.2. M. Kyo, K.Usui-Aoki, H. Koga, Anal. Chem, 2005, 77, 7115-7121.

Surface Chemistry

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,脂滴分析试剂盒深红色/1/LD06

Lipi probes are small molecule which emit strong fluorescence in hydrophobic environment such as in LDs.

Function of lipid droplets

脂滴 (LD) 由中性脂质组成，例如三酰基甘油和胆固醇酯，它们被磷脂单层包围，并且无处不在，不仅在脂肪细胞中 1)。 虽然 LDs 被简单地认为是一种脂质储存单元，但最近的一项研究表明，LDs 在调节脂质代谢、自噬2) 和细胞衰老3) 方面发挥着重要作用。因此，作为阐明其机制的重要工具，LDs 受到了极大的关注 LD的形成、生长、融合和收缩。

1) T. Fujimoto et al., “Lipid droplets: a classic organelle with new outfits.” Histochem Cell Biol., 2008, 130(2), 263.2) R. Singh et al., “Autophagy regulates lipid metabolism.” Nature, 2009, 458(7242), 1131.3) M. Yokoyama et al., “Inhibition of endothelial p53 improves metabolic abnormalities related to dietary obesity.” Cell Reports, 2014, 7(5), 1691.

Only by the addition of a reagent, the imaging of lipid droplets (LDs) or the quantitative variation of LDs in live and fixed cells becomes quantifiable.

Lipid Droplet Assay Kit 大大缩短了整个过程，可用于活细胞。Lipid Droplet Assay Kit 中提供的荧光染料可用于活细胞和固定细胞。 与使用比色试剂的方法相比，使用Lipid Droplet Assay Kit的方法可以缩短测量时间。 此外，使用 Lipid Droplet Assay Kit 可以提高实验的重复性，因为染料不会沉积在板中。

Experimental example of plate assayChanges in lipid droplets were examined after the addition of oleic acid or Triacsin C (acyl-CoA synthetase inhibitor) to the A549 cell culture medium.

As a result, we confirmed that the oleic acid-treated cells show an increase in the number of LDs, compared to control and Triacsin C-treated cells.

Blue　　 ：Ex. 376 – 386 nm / Em 435 – 455 nmDeep Red ：Ex. 623 – 633 nm / Em 649 – 669 nm

Reagent ComparisonChanges in lipid droplets were examined after the addition of oleic acid or Triacsin C (acyl-CoA synthetase inhibitor) to the HeLa cell culture medium.As a result, we confirmed that the oleic acid-treated cells show an increase in the number of LDs, compared to control and Triacsin C-treated cells.

<Detection Condition>Blue　　 ：Ex. 405 nm/ Em 425 – 475 nmDeep Red ：Ex. 640 nm/ Em 650 – 670 nm

Related Product Information

Function Product Code Product Size
Imaging LD01 Lipi-Blue 10 nmol
LD02 Lipi-Green 10 nmol
LD03 Lipi-Red 100 nmol
LD04 Lipi-Deep Red 10 nmol
Quantification (Plate Reader, FCM) LD05 Lipi Droplet Assay Kit-Blue 1 set
LD06 Lipi Droplet Assay Kit-Deep Red 1 set
Can I use Lipi series for fixed cells?

Yes, Lipi-dye can be used for fixed cells.* Please use paraformaldehyde (PFA) for fixation. Alcohol fixation is not recommended because it may affect the structure of lipid droplets.* Depending on the cell, it may not be stained or weakened in sensitivity due to fixed conditions before and after staining. In that case, please consider fixed conditions.○ Fix cells after staining1. Remove the medium and wash twice with PBS.2. Add Lipi series Working solution (in PBS) in the cells and incubated at 37 ℃ for 30 minutes.3. Remove the supernatant and wash twice with PBS.4. Add 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 5 minutes.5. Remove the supernatant and wash with PBS.○ Fix cells before staining1. Remove the medium and wash twice with PBS.2. Add 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 5 minutes.3. Remove the supernatant and wash twice with PBS.4. Add Lipi-dye Working solution(in PBS) in the cells and incubated at 37 ℃ for 15 minutes.5. Remove the supernatant and wash with PBS.

Preparing a stock solution of oleic acidRequired Reagents:・BSA (bovine serum albumin)・Oleic acid・0.1 mol/L Tris-HCl (pH 8.0)Procedure:(1) Dissolve 0.14 g/mL BSA in 0.1 mol/L Tris-HCl (pH 8.0).(2) Add 4 mmol/L oleic acid to a disposable centrifuge tube.(3) Add BSA solution (prepared in step 1).(4) Cap the tube and mix on a rotary shaker (Be sure the solution is transparent, indicating that oleic acid has been conjugated to BSA).(4) Filter the solution prepared above (step 4) using 0.22μm filter membranes.(5) Store oleic acid stock solution at 4˚C.*Use the appropriate amount of oleic acid stock solution for culture medium to prepare working solution.*Oleic acid working solution cannot be stored. Please prepare the working solution immediately before usage.

Inducing lipid droplets(1) Incubate cells for 24 hours at 37˚C in a 5% CO2 atmosphere.(2) Add 200 µmol/L working solution (prepared from oleic acid stock solution) to culture medium and incubate for further 24 hours.

Dojindo,10-羧基-1-癸硫醇/100/C385

羧基烷硫醇用于修饰金表面以在其上引入羧基。羧基通常转化为活化的 N-羟基琥珀酰亚胺酯，它与生物材料的胺基反应。 Dojindo 新开发的 15-Carboxy-1-pentadecanethiol 具有 15 个碳链，是市场上羧基烷硫醇中最长的烷硫醇。包括 Carboxy-EG6-十一烷硫醇在内的五种不同的羧基烷硫醇可用于金表面改性。 Malone 和其他人使用 15-Carboxy-1-pentadecanethiol 制造了一种高度灵敏的 SPR 传感器。 Glenn 和他的同事使用羧基烷硫醇和聚-L-赖氨酸来制造固定的细胞色素 b5 多层电极。 Mizutani 和其他人以类似的方式制造了固定化葡萄糖氧化酶多层电极。两组都报告了从生物材料到金表面的电子转移。这些类型的多层膜电极非常适用于扩散电子转移的研究。 Frisbie 和其他人开发了一种新方法，化学力显微镜，用于获得图案样品表面的粘合剂相互作用和摩擦图像。他们使用原子力显微镜 (AFM) 来测量化学上不同官能团的相互作用和空间映射。 Frisbie 和其他人在 AFM 悬臂尖端的金表面上形成了羧基烷硫醇单分子层。他们使用原子力显微镜测量分子修饰的探针尖端和有机单层之间的粘附力和摩擦力，这些有机单层以光刻定义的不同官能团图案终止。

1. J. D. H. Glenn and E. F. Bowden, Diffusionless Electrochemistry of Cytochrome b5 Adsorbed on a Multilayer Film Electrode, Chem. Lett., 1996, 399.2. F. Mizutani, Y. Sato, S. Yabuki and Y. Hirata, Enzyme Ultra-thin Layer Electrode Prepared by the Co-adsorption of Poly-L-lysine and Glucose Oxidase onto a Mercaptopropionic Acid-Modified Gold Surface, Chem. Lett., 1996, 251.3. C. D. Frisbie, F. Rozsnyai, A. Noy, M. S. Wrighton and C. M. Lieber, Functional Group Imaging by Chemical Force Microscopy, Science, 1994, 265,2071.4. M. Kyo, K. Usui-Aoki and H. Koga, Label-free Detection of Proteins in Crude Cell Lysate with Antibody Arrays by a Surface Plasmon Resonance Imaging Technique, Anal. Chem., 2005, 77, 7115.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,1-Methoxy PMS/100/M003

1-甲氧基 PMS 很容易被水和酒精溶解。 其氧化还原电位为+63 mV。 虽然甲基硫酸吩嗪鎓 (PMS) 通常用作 NADH-四唑鎓的电子载体，但 PMS 的稳定性很差。 但是，1-甲氧基 PMS 溶液可以在室温下储存 3 个月以上，无需避光。 NAD(P)H 的一个电子通过 1-甲氧基 PMS 转移到四唑盐（反应方案）。 因此，它是基于 NAD(P)H-四唑鎓测定系统的有用试剂。

1. R. Hisada, et al., 1-methoxy-5-methylphenazinium Methyl Sulfate. J Biochem. 1977;82:1469-1473.2. S. Nakamura, et al., Use of 1-methoxy-5-methylphenazinium Methyl Sulfate(1-methoxyPMS) in the Assay of Some Enzymes of Diagnosticimportance. Clin Chim Acta. 1980;101:321-326.3. C. J. Van Noorden, et al., The Role of Exogenous Electron Carriers in NAD(P)-dependent Dehydrogenase Cytochemistry Studied in vitro and with a Model System of Polyacrylamide Films. J Histochem Cytochem. 1982;30:12-20.4. H. Tsuge, et al., Partial Purification and Property of Pyridoxine (Pyridoxamine)-5 E phosphate Oxidase Isozymes from Wheat Seedlings. Arch Biochem Biophys. 1982;217:479-484.5. M. Rabinovitch, et al., Destruction of Leishmania Mexicana Amazonensis Amastigotes within Macrophages in Culture by Phenazine Methosulfate and Other Electron Carriers. J Exp Med. 1982;155:415-431.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,MADB/1/OC21

1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982;30:2492-2497.2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982;136:121-127.3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984;56:2849-2850.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,DTNB/1/D029

DTNB 被称为埃尔曼试剂。 它用于比色测定生物样品中的硫醇基团。 它相当易溶于水。 在硫醇化合物的存在下，无色 DTNB 转化为黄色 5-巯基-2-硝基苯甲酸（图 1）。 由于 5-Mercapto-2-nitrobenzoic acid 在 412 nm 处有最大吸收，DTNB 的吸收光谱不会干扰硫醇检测。

Fig. 1 DTNB reaction with thiol compound

Fig. 2 Absorption spectra of DTNB and reduced DTNB

1. D. R. Grassetti, et al., Determination of Sulfhydryl Groups with 2, 2 E or 4, 4 EDithiodipyridine. Arch Biochem Biophys. 1967;119:41-49.2. R. E. Humphrey, et al., Spectrophotometric Determination of Sulfite with 4, 4 EDithiodipyridine and 5, 5 EDithiobis-(2-nitrobenzoic acid). Anal Chem. 1970;42:698-702.3. R. E. Humphrey, et al., Spectrophotometric Determination of Cyanide with Organic Disulphides. Talanta. 1971;18:491-497.4. T. Uete, et al., Spectrophotometric Micromethod for Measuring Cholinesterase Activity in Serum or Plasma. Clin Chem. 1972;18:454-458.

Dojindo细胞分析

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

WST检测机制

 

ß-半乳糖苷酶检测试剂

细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢

应用 产品展示
细胞生长检测，药物筛选，比色/荧光检测 细胞计数试剂盒-8
细胞计数试剂盒8 + 96孔有机硅定向剂
细胞计数试剂盒-F
细胞毒性LDH检测试剂盒
-WST 96孔有机硅定向剂
MTT
了解检测机制的差异： 点击这里
细胞周期分析 细胞周期测定溶液深红色
细胞周期测定溶液蓝色

 

Dojindo,NOC 18/50/N379,NOC 是稳定的 NO-胺复合物

Product Description of NOC Compounds NOC 是稳定的 NO-胺复合物，可在生理条件下自发释放 NO，无需辅因子。 NO 释放速率取决于 NOC 的化学结构。与其他经典的 NO 供体（如硝酸甘油和 nitropurusside）相比，NOCs 自发生成 NO 的机制非常简单，并且副产物不会干扰细胞活动。一个 NOC 分子释放两个 NO 分子（如反应方案所示）；第二个NO分子的释放速度很慢。 NOC 可用于以受控速率将受控数量的纯 NO 添加到实验系统中，且副作用最小。通过改变 NOC 试剂的浓度和选择，可以轻松控制释放的 NO 量。 Dojindo 提供具有不同半衰期的四种不同的 NOC（NOC 5、7、12 和 18）。在碱性溶液（如 NaOH 水溶液）中制备的 NOC 储备溶液相对稳定。然而，NOC 原液应在一天内使用，因为它每天降解约 5%，即使在 -20ºC 下也是如此。在将储备溶液添加到样品溶液后立即开始释放 NO。

Nitric Oxide Release1. 使用 0.1 M NaOH 制备 10 mM NOC 储备溶液。 由于NOC原液不稳定，所以放在冰浴上一天用完。 2． 将适量的 NOC 储备溶液添加到要释放 NO 的样品溶液中。 为保持样品溶液的 pH 值，NOC 储备溶液的体积不应超过样品体积的 1/50。 样品溶液应具有足够的缓冲作用。 加入 NOC 储备溶液后，NO 将立即释放。Table 1 pH Dependency of NO Release at 37oC

1. K. Hayashi, et al., Action of Nitric Oxide as a Antioxidant Against Oxidation of Soybean Phosphatidylcholine Liposomal Membrane. FEBS Lett. 1995;370:37-40. (NOC 12)2. S. Shibuta, et al., Intracerebroventricular Administration of a Nitric Oxide-releasing Compound, NOC-18, Produces Thermal Hyperalgesia in Rats. Neurosci Lett. 1995;187:103-106. (NOC 18)3. S. Shibuta, et al., A new nitric oxide donor, NOC-18, exhibits a nociceptive effect in the rat formalin model. J Neurol Sci. 1996;141:1-5. (NOC 18)4. N. Yamanaka, et al., Nitric Oxide Released from Zwitterionic Polyamine/NO Adducts Inhibits Cu2+-induced Low Density Lipoprotein Oxidation. FEBS Lett. 1996;398:53-56. (NOC 5, NOC 7)5. D. Berendji, et al., Nitric Oxide Mediates Intracytoplasmic and Intranuclear Zinc Release. FEBS Lett. 1997;405:37-41.6. T. Ohnishi, et al., The Effect of Cu2+ on Rat Pulmonary Arterial Rings. Eur J Pharmacol. 1997;319:49-55. (NOC 7)7. Y. Adachi, et al., Renal Effect of a Nitric Oxide Donor, NOC 7, in Anethetized Rabbits. Eur J Pharmacol. 1997;324:223-226. (NOC 7)8. Y. Minamiyama, et al., Effect of Thiol Status on Nitric Oxide Metabolism in the Circulation. Arch Biochem Biophys. 1997;341:186-192. (NOC 7)
How do I prepare a stock solution?

Prepare 10-50 mM NOC solution with 0.1 M NaOH solution. Then add enough NOC solution to the cell culture to obtain a suitable concentration of NOC in cell culture. If the pH of the culture solution changes, use higher concentration of NOC.

What is the solubility of the NOC compounds?

NOC 5: 40 mg per 100 ml 0.1 M NaOH (2.2 M NOC 5)NOC 7: 70 mg per 100 ml 0.1 M NaOH (4.3 M NOC 7)NOC 12: 27 mg per 100 ml 0.1 M NaOH (1.5 M NOC 12)NOC 18: 20 mg per 100 ml 0.1 M NaOH (1.2 M NOC 18)

Is the stock solution stable?

The stock solution will lose 5% of its NOC activity per day, even when stored at -20oC. Please prepare fresh solution prior to use and keep the solution on an ice bath during the experiment. VI-1. Nitric Oxide Research: NO Donors

How is the half-life of NOC determined?

Prepare 20 mM NOC stock solution with 0.1 M NaOH. Warm PBS at 37oC. Add 100 ml NOC solution to 1.9 ml PBS. Using a UV spectrophotometer, immediately start measuring its absorbance at the maximum wavelength of the NOC. Continue measuring until no further spectra changes are observed.

Can I use NOC for in vivo experiments?

Yes. Please review the papers by Shibata and colleagues (1995, 1996).

Is the amount of NO released in vitro the same as in vivo?

The amount of NO released in the solution should be the same if the pH and temperature are the same. However, the activity of NO may be different in vivo because of other reactive components such as thiol compounds and heme.

Dojindo,ICG-EG4-Sulfo-OSu/1/I289,ICG 是一种荧光团

ICG 是一种荧光团，已在人类临床应用中使用了半个多世纪。 传统的活化 ICG 染料 ICG-Sulfo-OSu 已在体内成像研究中用作抗体偶联物。 然而，由于疏水特性，可见非特异性结合抗体（导致较高的背景）。 作为下一代 ICG 染料，合成具有短 PEG 接头的 ICG 以增加染料的亲水性。 聚乙二醇化的 ICG 显示出与抗体更高的共价结合，显着降低了非特异性结合。 因此，发现具有短 PEG 接头的 ICG 是更适合体内成像研究的化合物。

Structural Formula

Advantage of PEGylated ICGPEGylated ICG is more suitable for in vivo imaging due to low background.PEGylated ICG Derivatives (ICG-EGn-Sulfo-OSu)

Non-PEGylated ICG (ICG-Sulfo-Osu)

<Conjugation Ability>

<In vivo Background>

<Performance of PEGylated ICG>

<ICG-EG4, EG8 Sulfo-OSu>1.Sano, K.; Nakajima, T.; Miyazaki, K.; Ohuchi, Y.; Ikegami, T.; Choyke, L. P.; Kobayashi, H.; Bioconjugate Chem., 24, 811-816, (2013).<ICG-Sulfo-OSu>1. Ogawa, M.; Kosaka, N.; Choyke, L. P.; Kobayashi, H.; Cancer Res., 69, 1268 – 1272 (2009).2. Kosaka, N.; Ogawa, M.; Sato, N., Choyke, L.; P.; Kobayashi, H.; J Invest Dermatol, 129, 2818 E822 (2009).3. Kosaka N.; Ogawa, M.; Choyke, P. L.; Kobayashi H.; Future Oncol., 5, 1501-11 (2009).4. Nakajima T,; Mitsunaga M,; Bander NH,; Heston WD,; Choyke PL,; Kobayashi H.; Bioconjug Chem., 22,1700-1705 (2011).5. Ogawa, M.; Regino, C. A.; Seidel, J.; Green, M. V.; Xi, W.; Williams, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H.; Bioconjug Chem., 20, 2177-84 (2009).

Dojindo,NOC 7/50/N377,CompoundsNOC 是稳定的 NO-胺复合物

Product Description of NOC CompoundsNOC 是稳定的 NO-胺复合物，可在生理条件下自发释放 NO，无需辅因子。 NO 释放速率取决于 NOC 的化学结构。与其他经典的 NO 供体（如硝酸甘油和 nitropurusside）相比，NOCs 自发生成 NO 的机制非常简单，并且副产物不会干扰细胞活动。一个 NOC 分子释放两个 NO 分子（如反应方案所示）；第二个NO分子的释放速度很慢。 NOC 可用于以受控速率将受控数量的纯 NO 添加到实验系统中，且副作用最小。通过改变 NOC 试剂的浓度和选择，可以轻松控制释放的 NO 量。 Dojindo 提供具有不同半衰期的四种不同的 NOC（NOC 5、7、12 和 18）。在碱性溶液（如 NaOH 水溶液）中制备的 NOC 储备溶液相对稳定。然而，NOC 原液应在一天内使用，因为它每天降解约 5%，即使在 -20ºC 下也是如此。在将储备溶液添加到样品溶液后立即开始释放 NO。

Nitric Oxide Release1. 使用 0.1 M NaOH 制备 10 mM NOC 储备溶液。 由于NOC原液不稳定，所以放在冰浴上一天用完。 2． 将适量的 NOC 储备溶液添加到要释放 NO 的样品溶液中。 为保持样品溶液的 pH 值，NOC 储备溶液的体积不应超过样品体积的 1/50。 样品溶液应具有足够的缓冲作用。 加入 NOC 储备溶液后，NO 将立即释放。

Table 1 pH Dependency of NO Release at 37oC

1. K. Hayashi, et al., Action of Nitric Oxide as a Antioxidant Against Oxidation of Soybean Phosphatidylcholine Liposomal Membrane. FEBS Lett. 1995;370:37-40. (NOC 12)2. S. Shibuta, et al., Intracerebroventricular Administration of a Nitric Oxide-releasing Compound, NOC-18, Produces Thermal Hyperalgesia in Rats. Neurosci Lett. 1995;187:103-106. (NOC 18)3. S. Shibuta, et al., A new nitric oxide donor, NOC-18, exhibits a nociceptive effect in the rat formalin model. J Neurol Sci. 1996;141:1-5. (NOC 18)4. N. Yamanaka, et al., Nitric Oxide Released from Zwitterionic Polyamine/NO Adducts Inhibits Cu2+-induced Low Density Lipoprotein Oxidation. FEBS Lett. 1996;398:53-56. (NOC 5, NOC 7)5. D. Berendji, et al., Nitric Oxide Mediates Intracytoplasmic and Intranuclear Zinc Release. FEBS Lett. 1997;405:37-41.6. T. Ohnishi, et al., The Effect of Cu2+ on Rat Pulmonary Arterial Rings. Eur J Pharmacol. 1997;319:49-55. (NOC 7)7. Y. Adachi, et al., Renal Effect of a Nitric Oxide Donor, NOC 7, in Anethetized Rabbits. Eur J Pharmacol. 1997;324:223-226. (NOC 7)8. Y. Minamiyama, et al., Effect of Thiol Status on Nitric Oxide Metabolism in the Circulation. Arch Biochem Biophys. 1997;341:186-192. (NOC 7)
How do I prepare a stock solution?

Prepare 10-50 mM NOC solution with 0.1 M NaOH solution. Then add enough NOC solution to the cell culture to obtain a suitable concentration of NOC in cell culture. If the pH of the culture solution changes, use higher concentration of NOC.

What is the solubility of the NOC compounds?

NOC 5: 40 mg per 100 ml 0.1 M NaOH (2.2 M NOC 5)NOC 7: 70 mg per 100 ml 0.1 M NaOH (4.3 M NOC 7)NOC 12: 27 mg per 100 ml 0.1 M NaOH (1.5 M NOC 12)NOC 18: 20 mg per 100 ml 0.1 M NaOH (1.2 M NOC 18)

Is the stock solution stable?

The stock solution will lose 5% of its NOC activity per day, even when stored at -20oC. Please prepare fresh solution prior to use and keep the solution on an ice bath during the experiment. VI-1. Nitric Oxide Research: NO Donors

How is the half-life of NOC determined?

Prepare 20 mM NOC stock solution with 0.1 M NaOH. Warm PBS at 37ºC. Add 100 ml NOC solution to 1.9 ml PBS. Using a UV spectrophotometer, immediately start measuring its absorbance at the maximum wavelength of the NOC. Continue measuring until no further spectra changes are observed.

Can I use NOC for in vivo experiments?

Yes. Please review the papers by Shibata and colleagues (1995, 1996).

Is the amount of NO released in vitro the same as in vivo?

The amount of NO released in the solution should be the same if the pH and temperature are the same. However, the activity of NO may be different in vivo because of other reactive components such as thiol compounds and heme.

Dojindo,NOR 5/10/N448,NORs 是理想的 NO 供体

Product Description of NOR CompoundsNORs 是理想的 NO 供体，其化学结构与其他 NO 供体完全不同。尽管 NOR 没有任何 ONO2 或 ONO 部分，但它们会以稳定的速率自发释放 NO。尽管 NOR 的 NO 释放机制尚未完全确定，但已证实其副产物不具有任何显着的生物活性。据报道，从 Streptomyces genseosporeus 中分离出的 NOR 3 对大鼠和兔主动脉和狗冠状动脉具有强烈的血管舒张作用。其活性（ED50=1 nM）是硝酸异山梨酯（ISDN）的300倍。 NOR 3 也增加血浆循环 GMP 水平，而 ISDN 没有。 NOR 是血小板聚集和血栓形成的有效抑制剂。 NOR 3 (IC50=0-7 mM) 有效抑制 100% ADP 引发的人血小板聚集，而 ISDN 仅抑制总聚集的 32%，即使在 100 mM 浓度下也是如此。据报道，NOR 3 在缺血/再灌注系统中具有抗心绞痛和心脏保护作用。在大鼠乙酰甲胆碱诱导的冠状血管痉挛模型中，NOR 3 在 1 mg/kg 时剂量依赖性地显着抑制 ST 段的升高。另一方面，ISDN 将其显着抑制为每公斤 3.2 毫克。 NOR试剂的NO释放率的差异甚至反映在体内降血压作用上。 NOR 也可以在 0.5% 甲基纤维素悬浮液中口服使用。 NOR在DMSO溶液中相对稳定。 NOR 1 具有最短的半衰期，是一种很有前途的试剂，可用于制备用于校准的 NO 标准溶液。为了制备标准溶液，将精确稀释的 NOR 1/DMSO 溶液添加到缓冲溶液中。

Nitric Oxide Release1. 使用 DMSO 制备 10 mM NOR 储备溶液。 由于 NOR 原液不稳定，请将其放在冰浴上，并在一天内使用。 2． 将适量的 NOR 储备溶液添加到要释放 NO 的样品溶液中。 为避免 DMSO 可能对细胞造成损害，NOR 原液的体积不应超过样品体积的 1/50。 样品溶液应具有足够的缓冲作用。 加入 NOR 储备溶液后，NO 将立即释放。

1. S. Shibata, et al., Characteristics of the Vasorelaxing Action of (3E)-4-Ethyl-2-hydroxyimino-5-nitro-3-hexamide FK409, a New Vasodilator Isolated from Microbial Sources, in Isolated Rabbit Arteries. J Cardiovasc Pharmacol. 1991;17:508-518.2. Y. Kita, et al., Antianginal Effects of FK409, a New Spontaneous NO Releaser. Br J Pharmacol. 1994;113:1137-1140.3. Y. Kita, et al., Antiplatelet Activities of FK409, a New Spontaneous NO Releaser. Br J Pharmacol. 1994;113:385-388.4. Y. Kita, et al., Spontaneous Nitric Oxide Release Accounts for the Potent Pharmacological Actions of FK409. Eur J Pharmacol. 1994;257:123-130.5. M. Hino, et al., FK409, a Novel Vasodilator Isolated from the Acid-treated Fermentation Broth of Streptomyces Griseosporeus I. Taxonomy, Fermentation, Isolation, and Physico-chemical and Biological Characteristics. J Antibiot. 1989; 42:1578-1583.6. J. Decout, et al., Decomposition of FK409, a New Vasodilator: Identification of Nitric Oxide as Metaborite. Bioorg Med Chem Lett. 1995;5:973-978.7. S. Fukuyama, et al., A New Nitric Oxide (NO) Releaser: Spontaneous NO Release from FK409. Free Radic Res. 1995;23:443-452.8. Y. Kita, et al., FR144420, a Novel, Slow, Nitric Oxide-releasing Agent. Eur J Pharmacol. 1995;275:125-130.9. M. Kato, et al., New Reagents for Controlled Release of Nitric Oxide. Structure-stability Relationships. Bioorg Med Chem Lett. 1996;6:33-38.10. Y. Kita, et al., FK409, a Novel Spontaneous NO Releaser: Comparative Pharmacological Studies with ISDN. Cardiovasc Drug Rev. 1996;14:148-165.11. Y. Hirasawa, et al., Antianginal Effects of FR144420, a Novel Nitric Oxide-releasing Agent. Eur J Pharmacol. 1996;303:55-59.12. M. Sato, et al., Nitric Oxide Raises Cytosolic Concentrations of Ca2+ in Cultured Nodose Ganglion Neurons from Rabbits. Neurosci Lett. 1996;206:69-72.13. Y. Kita, et al., Oral Biological Activities of Spontaneous Nitric Oxide Releaser are Accounted for by their Nitric Oxide-releaseing Rates and Oral Absorption Manners. J Pharmacol Exp Ther. 1996;276:421-425.14. S. Fukuyama, et al., Structure-activity Relationships of Spontaneous Nitric Oxide Releasers, FK409 and its Derivatives. J Pharmacol Exp Ther. 1997;282:236-242.15. Y. Kita, et al., Comparison of Hemodynamic Effects of Nitric Oxide (NO) Donors with Different NO-releasing Properties in Rats. J Cardiovasc Pharmacol. 1997;30:223-228.16. Y. Hirasawa, et al., Comparison of Antiplatelet Effect of two Nitric Oxide-donating Agents, FR146801 and FK409. Thromb Haemost. 1998;79:620-624.
How do I prepare a stock solution?

用 DMSO 制备 10-50 mM NOR 溶液。 DMSO 应干燥。 然后向细胞培养物中加入足够的 NOR 溶液，以获得合适浓度的 NOR。

What is the solubility of NOR compounds?

NOR 1: 100 mg per 100 ml DMSO (4.3 M)NOR 3: 137 mg per 100 ml DMSO (6.4 M)NOR 4: 30 mg per 100 ml DMSO (1.0 M)NOR 5: 30 mg per 100 ml DMSO (0.9 M)

Is oral administration possible?

Yes. Please review the article by Kita and colleagues (Eur. J. Pharmacol., 257, 123-130, 1994).

How many NO molecules does each NOR molecule release in physiological conditions? What are the byproducts?

在生理条件下，平均每个 NOR 分子释放 1 到 1.5 个 NO 分子。 不幸的是，NOR副产物的结构仍不清楚。 然而，对于 NO 释放实验，NOR 副产物在正常浓度下没有细胞毒性。

Dojindo,FeBABE/1/F279,Bromoacetamidobenzyl-EDTA (BABE) 是一种螯合标记试剂与巯基结合

Product Description of BABEs
Bromoacetamidobenzyl-EDTA (BABE) 是一种螯合标记试剂，可与巯基结合。 BABE 的铁螯合物 (FeBABE) 是一种独特的工具，用于确定蛋白质的三维结构以及蛋白质-蛋白质或蛋白质-DNA 复合物的结合结构。 BABE 通过其巯基将 EDTA 部分添加到蛋白质中。 一旦附着在蛋白质上，FeBABE 就会切割附近的肽或 DNA 链。 切割位点在 FeBABE 结合位点的 12 埃以内。 铁 (II)-螯合物在过氧化氢存在下切割肽或 DNA 链。 裂解反应迅速完成：10 秒到 20 分钟的孵育就足够了。 用凝胶电泳如 SDS-PAGE 分析切割片段的大小。
Structural Formula:

Labeling Procedure1. 将蛋白溶液在偶联缓冲液（10-20 mM MOPS、0.2 M NaCl、2 mM EDTA、5% 甘油、pH 8.0）中于 4ºC 透析过夜。 2. 透析后，将蛋白质浓度调整到 15-30 mM.3。 将 15 μl 20 mM FeBABE DMSO 溶液添加到 1 ml 蛋白质溶液中，并在 37ºC 下孵育 1 小时。 FeBABE 的最终浓度为 0.3 mM（比蛋白质过量 10-20 倍）。4。 在 4ºC 下将反应混合物在蛋白质储存缓冲液（10-20 mM Tris、0.1-0.2 M KCl、10 mM MgCl2、0.1 mM EDTA、50% 甘油、pH 7.6）中透析过夜。

Amrita Kumar and Charles P. Moran, Jr., Promoter Activation by Repositioning of RNA Polymerase, JOURNAL OF BACTERIOLOGY, 2008, 190, 3110.Erin L. Benanti1 and Peter T. Chivers, Helicobacter pylori NikR Protein Exhibits Distinct Conformations When Bound to Different Promoters, THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2011, 286, 15728.

Chih-Chien Wu, Yu-Chun Lin, and Hung-Ta Chen, The TFIIF-Like Rpc37/53 Dimer Lies at the Center of a Protein Network To Connect TFIIIC, Bdp1, and the RNA Polymerase III Active Center, MOLECULAR AND CELLULAR BIOLOGY, 2011, 31, 2715.

1. L. H. DeRiemer, C. F. Meares, D. A. Goodwin and C. I. Diamanti, BLEDTA II: Synthesis of a New Tumer-Visualizing Derivative of Co(III)-bleomycin, J. Labelled Compd. Radiopharm., 1981, 18, 1517.2. T. M. Rana and C. F. Meares, Specific Cleavage of a Protein by an Attached Iron Chelate, J. Am. Chem. Soc., 1990, 112, 2457.3. T. M. Rana and C. F. Meares, Transfer of Oxigen from an artificial protease to peptide carbon during proteolysis, Proc. Natl. Acad. Sci. USA, 1991, 88, 10578.4. D. P. Greiner, R. Miyake, J. K. Moran, A. D. Jones, T. Negishi, A. Ishihama, and C. F. Meares, Synthesis of the Protein Cutting Reagent Iron (S)-1-(p- Bromoacetamidobenzyl) ethylebediaminetetraacetate and Conjuation to cysteine Sie Cahins, Bioconjugate Chem., 1997, 8, 44.5. E. Platis, M. R. Ermacora and R. O. Fox, Oxidative Polypeptide Cleavage Mediated by EDTA-Fe Covalently Linked to Cysteine Residue, Biochemistry, 1993, 32, 12761.6. S. L. Traviglia, S. A. Datwyler, D. Yan, A. Ishihama and C. F. Meares, Targeted Protein Footprinting: Where Different Transcription Factors bind to RNA Polymerase, Biochemistry, 1999, 38, 4259.7. J. B. Ghaim, D. P. Greiner, C. F. Meares and R. B. Gennis, Proximity Mapping the suface of Membrane Protein Using an Artificial Protease: Demonstration That the Quinone-Binding Domain of Subunit I Is near the N-Terminal Region of Subunit II of Cytochrome bd, Biochemistry, 1995, 34, 11311.8. R. Miyake, K. Murakami, J. T. Owens, D. P. Greiner, O. N. Ozoline, A. Ishihama and C. F. Meares, Dimeric Association of Escherichia coli RNA Polymerase alfa subunits, studied by Cleavage of Single-Cysteine alfa Sununits Conjugated to Iron-(S)-1-(p-(Bromoacetamido)benzyl) ethylenediaminetetraacetate, Biochemistry, 1998, 37, 1344.9. J. T. Owens, R. Miyake, K. Murakami, A. J. Chmura, N. Fujita, A. Ishihama and C. F. Meares, Mapping the sigma70 subunits contact sites on Escherichia coli RNA polymerase with a sigma70-conjugated chemical protease, Proc. Natl. Acad. Sci. USA, 1998, 95, 6021.10. J. A. Bown, J. T. Owens, C. F. Meares, N. Fujita, A. Ishihama, S. J. Busby and S. D. Minchin, Organization of open complexes at Escherichia coli promoters. Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase, J. Biol. Chem., 1999, 274, 2263.11. F. Colland, N. Fujita, D. Kotlarz, J. A. Bown, C. F. Meares, A. Ishihama and A. Kolb, Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes, EMBO J., 1999, 18, 4049.12. G. M. Heilek, R. Marusak, C. F. Meares and H. F. Noller, Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4, Proc. Natl. Acad. Sci. USA, 1995, 92, 1113.13. G. M. Heilek and H. F. Noller, Site-directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S5, Science, 1996, 272, 1659.14. K. R. Lieberman and H. F. Noller, Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure., J. Mol. Biol., 1998, 284, 1367.

Dojindo,2-PDS/1/P016

Dojindo,2-PDS/1/P016
2-PDS reaction with thiol compound

1. D. R. Grassetti, et al., Determination of Sulfhydryl Groups with 2, 2 E or 4, 4 EDithiodipyridine. Arch Biochem Biophys. 1967;119:41-49.2. R. E. Humphrey, et al., Spectrophotometric Determination of Sulfite with 4, 4 EDithiodipyridine and 5, 5 EDithiobis-(2-nitrobenzoic acid). Anal Chem. 1970;42:698-702.3. R. E. Humphrey, et al., Spectrophotometric Determination of Cyanide with Organic Disulphides. Talanta. 1971;18:491-497.4. T. Uete, et al., Spectrophotometric Micromethod for Measuring Cholinesterase Activity in Serum or Plasma. Clin Chem. 1972;18:454-458.

Dojindo,NOC 12/10/N378,NOC 是稳定的 NO-胺复合物

Product Description of NOC Compounds

NOC 是稳定的 NO-胺复合物，可在生理条件下自发释放 NO，无需辅因子。 NO 释放速率取决于 NOC 的化学结构。与其他经典的 NO 供体（如硝酸甘油和 nitropurusside）相比，NOCs 自发生成 NO 的机制非常简单，并且副产物不会干扰细胞活动。一个 NOC 分子释放两个 NO 分子（如反应方案所示）；第二个NO分子的释放速度很慢。 NOC 可用于以受控速率将受控数量的纯 NO 添加到实验系统中，且副作用最小。通过改变 NOC 试剂的浓度和选择，可以轻松控制释放的 NO 量。 Dojindo 提供具有不同半衰期的四种不同的 NOC（NOC 5、7、12 和 18）。在碱性溶液（如 NaOH 水溶液）中制备的 NOC 储备溶液相对稳定。然而，NOC 原液应在一天内使用，因为它每天降解约 5%，即使在 -20ºC 下也是如此。在将储备溶液添加到样品溶液后立即开始释放 NO。

Nitric Oxide Release1. 使用 0.1 M NaOH 制备 10 mM NOC 储备溶液。 由于NOC原液不稳定，所以放在冰浴上一天用完。 2． 将适量的 NOC 储备溶液添加到要释放 NO 的样品溶液中。 为保持样品溶液的 pH 值，NOC 储备溶液的体积不应超过样品体积的 1/50。 样品溶液应具有足够的缓冲作用。 加入 NOC 储备溶液后，NO 将立即释放。Table 1 pH Dependency of NO Release at 37oC

1. K. Hayashi, et al., Action of Nitric Oxide as a Antioxidant Against Oxidation of Soybean Phosphatidylcholine Liposomal Membrane. FEBS Lett. 1995;370:37-40. (NOC 12)2. S. Shibuta, et al., Intracerebroventricular Administration of a Nitric Oxide-releasing Compound, NOC-18, Produces Thermal Hyperalgesia in Rats. Neurosci Lett. 1995;187:103-106. (NOC 18)3. S. Shibuta, et al., A new nitric oxide donor, NOC-18, exhibits a nociceptive effect in the rat formalin model. J Neurol Sci. 1996;141:1-5. (NOC 18)4. N. Yamanaka, et al., Nitric Oxide Released from Zwitterionic Polyamine/NO Adducts Inhibits Cu2+-induced Low Density Lipoprotein Oxidation. FEBS Lett. 1996;398:53-56. (NOC 5, NOC 7)5. D. Berendji, et al., Nitric Oxide Mediates Intracytoplasmic and Intranuclear Zinc Release. FEBS Lett. 1997;405:37-41.6. T. Ohnishi, et al., The Effect of Cu2+ on Rat Pulmonary Arterial Rings. Eur J Pharmacol. 1997;319:49-55. (NOC 7)7. Y. Adachi, et al., Renal Effect of a Nitric Oxide Donor, NOC 7, in Anethetized Rabbits. Eur J Pharmacol. 1997;324:223-226. (NOC 7)8. Y. Minamiyama, et al., Effect of Thiol Status on Nitric Oxide Metabolism in the Circulation. Arch Biochem Biophys. 1997;341:186-192. (NOC 7)
How do I prepare a stock solution?

Prepare 10-50 mM NOC solution with 0.1 M NaOH solution. Then add enough NOC solution to the cell culture to obtain a suitable concentration of NOC in cell culture. If the pH of the culture solution changes, use higher concentration of NOC.

What is the solubility of the NOC compounds?

NOC 5: 40 mg per 100 ml 0.1 M NaOH (2.2 M NOC 5)NOC 7: 70 mg per 100 ml 0.1 M NaOH (4.3 M NOC 7)NOC 12: 27 mg per 100 ml 0.1 M NaOH (1.5 M NOC 12)NOC 18: 20 mg per 100 ml 0.1 M NaOH (1.2 M NOC 18)

Is the stock solution stable?

即使储存在 -20oC，储备溶液每天也会失去 5% 的 NOC 活性。 请在使用前准备新鲜溶液，并在实验过程中将溶液置于冰浴中。 六一1。 一氧化氮研究：没有供体

How is the half-life of NOC determined?

用 0.1 M NaOH 制备 20 mM NOC 库存溶液。 在 37oC 下加热 PBS。 在 1.9 ml PBS 中加入 100 ml NOC 溶液。 使用紫外分光光度计，立即开始测量其在 NOC 最大波长处的吸光度。 继续测量，直到没有观察到进一步的光谱变化。

Can I use NOC for in vivo experiments?

Yes. Please review the papers by Shibata and colleagues (1995, 1996).

Is the amount of NO released in vitro the same as in vivo?

如果 pH 值和温度相同，则溶液中释放的 NO 量应相同。 然而，由于硫醇化合物和血红素等其他反应性成分，NO 在体内的活性可能有所不同。

Dojindo,MDB/100/M021,苯二胺 (OPD) 与 α-酮酸反应形成高荧光喹喔啉衍生物

Product Description

苯二胺 (OPD) 与 α-酮酸反应形成高荧光喹喔啉衍生物。 MDB 是邻苯二胺类似物。 由于它的反应性和敏感性，它是α-酮酸的最佳标记试剂。 在 HPLC 分析中，即使每次注射的浓度仅为几飞摩尔，也可以一次分别检测出十多种不同的 α-酮酸，例如 α-酮戊二酸、丙酮酸和对羟基苯丙酮酸。 MDB 的灵敏度几乎是 OPD 的 150 倍。 标记材料的激发和发射波长分别为 367 nm 和 445 nm。

1. M. Nakamura, et al., 1, 2-Diamino-4, 5-methylenedioxybenzene as a Highly Sensitive Fluorogenic Reagent for α-Keto Acids. Chem Pharm Bull. 1987;35:687-692.2. S. Hara, et al., Fluorometric High-Performance Liquid Chromatography of N-Acetyl-and N-Glycolylneuraminic Acids and Its Application to Their Microdetermination in Human and Animal Sera, Glycoproteins, and Glycolipids. Anal Biochem. 1987;164:138-145.3. S. Hara, et al., 1, 2-Diamino-4, 5-methylenedioxybenzene as a Highly Sensitive Fluorogenic Reagent for α-Dicarbonyl Compounds. Anal Chim Acta. 1988;215:267-276.

Dojindo,HiLyte Fluor 555标记试剂盒-NH2/3/LK14

HiLyte Fluor* 555 Labeling Kit-NH2 主要用于制备红色荧光标记的蛋白质，例如用于免疫染色的 IgG，以及用于示踪的细胞蛋白质。 NH2 反应性 HiLyteFluor 555 是该试剂盒的一个组成部分，具有与蛋白质或其他分子上的氨基反应的琥珀酰亚胺基 (NHS)（图 1）。 该试剂盒包含标记所需的所有试剂。 每管 HiLyte Fluor 555 最多可标记 200 μg 的 IgG，每个 IgG 分子可结合约 4 至 6 个 HiLyte Fluor 555 分子。 标记过程很简单——将 NH2 反应性 HiLyte Fluor 555 添加到膜上的 IgG 溶液中，并在 37ºC 下孵育 10 分钟。 多余的 HiLyte Fluor 555 分子可以通过过滤管去除。 HiLyte Fluor 555 标记的 IgG 的激发和发射波长分别为 555 nm 和 570 nm（图 2）。* HiLyte Fluor 是 AnaSpec, Inc. 的商标。

Fig. 1 Fluorescence Spectrum of HiLyte Fluor 555-conjugated IgG

Fig. 2 Fluorescence spectrum of HiLyte Fluor 555-conjugated IgG______excitation spectrum______emission spectrum

Precaution♦ 本试剂盒要标记的蛋白质的分子量应大于 50,000。♦ 标记过程中，IgG 或 HiLyte Fluor 555 结合的 IgG 始终在过滤管的膜上。♦ 如果 IgG 溶液中含有其他蛋白质 分子量大于 10,000（例如 BSA 或明胶）的 IgG 溶液，在使用该试剂盒标记 HiLyte Fluor 555 之前纯化 IgG 溶液。 IgG 溶液可通过 IgG 纯化试剂盒（本试剂盒未包含）进行纯化。♦ 如果 IgG 溶液含有少量不溶性物质，则将溶液离心并使用上清液进行标记。

1. A. Nagao, K. Sato, K.M. Nishida, H. Siomi, M.C. Siomi, “Gender-specific hierarchy in nuage localization of PIWI-interacting RNA factors in Drosophila”, Front Genet., 2011, 2, 55.2. H. Hoshino, G. Shioi, S. Aizawa, “AVE protein expression and visceral endoderm cell behavior during anterior–posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression”, Dev. Biol.., 2015, 402, (2), 175.3. T. Kaneko, T. Minohara, S. Shima, K. Yoshida, A. Fukuda, N. Iwamori, T. Inai and H. Iida, “A membrane protein, TMCO5A, has a close relationship with manchette microtubules in rat spermatids during spermiogenesis”, Mol. Reprod. Dev.., 2019, 86, (3), 330.4. Y. Nakashima, T. Mima, M. Yashiro, T. Sonou, M. Ohya, A. Masumoto, S. Yamanaka, D. Koreeda, K. Tatsuta, Y. Hanba, M. Moribata, S. Negi, T. Shigematsu, “Expression and localization of fibroblast growth factor (FGF) 23 and Klotho in the spleen: its physiological and functional implications”, Growth Factors ., 2016, 34, (5-6), 196.5. Z. Zhang, K. Kakutani, K. Maeno, T. Takada, T. Yurube, M. Doita, M. Kurosaka and K. Nishida, “Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis”, Arthritis Res. Ther.., 2011, 13, (6), R200.

Can I use this kit for other proteins?

是的，如果分子量大于 50,000。

在标记蛋白质之前是否必须使用过滤管？
如果蛋白质溶液不含带氨基的小分子，蛋白质浓度为10 mg/ml，或约70 μM，则无需使用过滤管。 将 10 μl 样品溶液与 90 μl 反应缓冲液混合，然后加入 8 μl NH2 反应性 HiLyte Fluor 555（在步骤 3 中制备）至

How long is the HiLyte Fluor 555-labeled protein stable?

如果储存在 4oC，它可以稳定超过 2 个月。 如需更长的储存时间，请加入 100% 体积的甘油，分装并储存在 -20oC。 但是，请注意稳定性取决于蛋白质本身。

该试剂盒可以标记的最少 IgG 量是多少？
IgG 的最小量为 10 μg。 对于少于 20 μg，按照手册添加 4 μl NH2 反应性 HiLyte Fluor 555，而不是在第 4 步中添加 8 μl。

Can I use this kit to label oligonucleotides or peptides?

不可以。寡核苷酸和肽可能太小而无法保留在过滤管的膜过滤器上。

Dojindo,Allophycocyanin Labeling Kit-NH2/3/LK21,别藻蓝蛋白标记试剂盒

藻胆蛋白是源自蓝细菌和真核藻类的荧光蛋白。 它们的荧光远高于荧光素和罗丹明等化学荧光探针。 别藻蓝蛋白 (APC) 是一种藻胆蛋白，在 660 nm 附近有红色荧光（图 1）。 由于这种高荧光，藻胆蛋白标记的抗体和其他分子可以在流式细胞术和免疫染色中提供更高的灵敏度。 别藻蓝蛋白标记试剂盒-NH2 用于简单快速地制备 APC 标记的 IgG（图 2）。 NH2-reactive APC（本试剂盒的一个组成部分）具有活化的酯基，无需任何活化过程即可轻松与目标分子的氨基形成共价键。 该试剂盒中的过滤管用于缓冲液交换和样品 IgG 溶液的浓缩。 该试剂盒包含 APC 标记所需的所有试剂，包括偶联物的储存缓冲液。
 Fluorescence spectrum of APCExcitation wavelength: 650 nmEmission wavelength: 660 nm
IgG labeling reaction of NH2-reactive APC

Precaution♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.♦ IgG or allophycocyanin-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with molecular weight greater than 10,000, such as BSA or gelatin, purify the IgG solution before labeling allophycocyanin with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

1. H. Shinohara, T. Yasuda, Y. Aiba, H. Sanjo, M. Hamadate, H. Watarai, H. Sakurai and T. Kurosaki, “PKCβ Regulates BCR-mediated IKK Activatioin by Facilitating the Interaction between TAK1 and CARMA1”, J. Exp. Med., 2005, 202(10), 1423.2. E. Grace Suto, Y. Mabuchi, N. Suzuki, K. Suzuki, Y. Ogata, M. Taguchi, T. Muneta, I. Sekiya and C. Akazawa, “Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation”, Sci. Rep.., 2017,(7), 4838.3. H. Fujii, Y. Ikeuchi, Y. Kurata, N. Ikeda, U. Bahrudin, P. Li, Y. Nakayama, R. Endo, A. Hasegawa, K. Morikawa, J. Miake, A. Yoshida, K. Hidaka, T. Morisaki, H. Ninomiya, Y. Shirayoshi, K. Yamamoto, I. Hisatome, “Electrophysiological properties of prion-positive cardiac progenitors derived from murine embryonic stem cells”, Circ. J.., 2012, 76, (12), 2875.

Can I use this kit for other proteins?
Yes, if the molecular weight is higher than 50,000 and it has a reactiveprimary or secondary amino group. Follow the protocol for IgG labeling with 0.5-1 nmol of sample protein.
How many APC molecules per IgG are introduced?
The average number of APC molecule per IgG is 1 to 2.
Does unconjugated NH2-reactive APC still have an activated ester after the labeling reaction to IgG??
No. It is completely hydrolyzed during the reaction.
Does NH2-reactive APC form an oligomer during the labeling reaction?
No. Since all amino groups of NH2-reactive APC are blocked, no oligomerization is possible.
What is the minimum amount of IgG that can be labeled with LK21-10?
The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG.
Do I have to use the WS buffer included with the kit?
Yes. Use the WS buffer to prepare a stock solution of the conjugate. However, you can choose any kind of buffer appropriate to dilute the conjugate stock solution for your experiment.
How long is the conjugate stable?
If you store at 4ºC, it is stable for over 2 months. For longer storage, add 100% volume of glycerol, aliquot and store at -20ºC. However, please note that the stability depends on the protein itself.

Dojindo,ICG-EG8-Sulfo-OSu/1/I290,ICG 是一种荧光团

ICG 是一种荧光团，已在人类临床应用中使用了半个多世纪。 传统的活化 ICG 染料 ICG-Sulfo-OSu 已在体内成像研究中用作抗体偶联物。 然而，由于疏水特性，可见非特异性结合抗体（导致较高的背景）。 作为下一代 ICG 染料，合成了具有短 PEG 接头如 ICG-PEG4-Sulfo-OSu 和 ICG-PEG8-SUlfo-OSu 的 ICG，以增加染料的亲水性。 聚乙二醇化的 ICG 显示出与抗体更高的共价结合，显着降低了非特异性结合。 因此，发现具有短 PEG 接头的 ICG 是更适合体内成像研究的化合物。

Structural Formula

Advantage of PEGylated ICGPEGylated ICG is more suitable for in vivo imaging due to low background.

PEGylated ICG Derivatives (ICG-EGn-Sulfo-OSu)

Non-PEGylated ICG (ICG-Sulfo-Osu)

<Conjugation Ability>

<In vivo Background>

<Performance of PEGylated ICG>

<ICG-EG4, EG8 Sulfo-OSu>1.Sano, K.; Nakajima, T.; Miyazaki, K.; Ohuchi, Y.; Ikegami, T.; Choyke, L. P.; Kobayashi, H.; Bioconjugate Chem., 24, 811-816, (2013).<ICG-Sulfo-OSu>1. Ogawa, M.; Kosaka, N.; Choyke, L. P.; Kobayashi, H.; Cancer Res., 69, 1268 – 1272 (2009).2. Kosaka, N.; Ogawa, M.; Sato, N., Choyke, L.; P.; Kobayashi, H.; J Invest Dermatol, 129, 2818 E822 (2009).3. Kosaka N.; Ogawa, M.; Choyke, P. L.; Kobayashi H.; Future Oncol., 5, 1501-11 (2009).4. Nakajima T,; Mitsunaga M,; Bander NH,; Heston WD,; Choyke PL,; Kobayashi H.; Bioconjug Chem., 22,1700-1705 (2011).5. Ogawa, M.; Regino, C. A.; Seidel, J.; Green, M. V.; Xi, W.; Williams, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H.; Bioconjug Chem., 20, 2177-84 (2009).

Dojindo,诺尔3/10/N390,NORs 是理想的 NO 供体

Product Description of NOR Compounds

NORs 是理想的 NO 供体，其化学结构与其他 NO 供体完全不同。尽管 NOR 没有任何 ONO2 或 ONO 部分，但它们会以稳定的速率自发释放 NO。尽管 NOR 的 NO 释放机制尚未完全确定，但已证实其副产物不具有任何显着的生物活性。据报道，从 Streptomyces genseosporeus 中分离出的 NOR 3 对大鼠和兔主动脉和狗冠状动脉具有强烈的血管舒张作用。其活性（ED50=1 nM）是硝酸异山梨酯（ISDN）的300倍。 NOR 3 也增加血浆循环 GMP 水平，而 ISDN 没有。 NOR 是血小板聚集和血栓形成的有效抑制剂。 NOR 3 (IC50=0-7 mM) 有效抑制 100% ADP 引发的人血小板聚集，而 ISDN 仅抑制总聚集的 32%，即使在 100 mM 浓度下也是如此。据报道，NOR 3 在缺血/再灌注系统中具有抗心绞痛和心脏保护作用。在大鼠乙酰甲胆碱诱导的冠状血管痉挛模型中，NOR 3 在 1 mg/kg 时剂量依赖性地显着抑制 ST 段的升高。另一方面，ISDN 将其显着抑制为每公斤 3.2 毫克。 NOR试剂的NO释放率的差异甚至反映在体内降血压作用上。 NOR 也可以在 0.5% 甲基纤维素悬浮液中口服使用。 NOR在DMSO溶液中相对稳定。 NOR 1 具有最短的半衰期，是一种很有前途的试剂，可用于制备用于校准的 NO 标准溶液。为了制备标准溶液，将精确稀释的 NOR 1/DMSO 溶液添加到缓冲溶液中。

Nitric Oxide Release1. Prepare 10 mM NOR stock solution using DMSO. Since the NOR stock solution is not stable, keep it on an ice bath and use it in one day.2. Add an appropriate volume of the NOR stock solution to the sample solution in which NO is to be released. In order to avoid possible damage to cells by DMSO, the volume of the NOR stock solution should not exceed 1/50 of the sample volume. The sample solution should have sufficient buffering action. NO will be released immediately after the addition of the NOR stock solution.
1. S. Shibata, et al., Characteristics of the Vasorelaxing Action of (3E)-4-Ethyl-2-hydroxyimino-5-nitro-3-hexamide FK409, a New Vasodilator Isolated from Microbial Sources, in Isolated Rabbit Arteries. J Cardiovasc Pharmacol. 1991;17:508-518.2. Y. Kita, et al., Antianginal Effects of FK409, a New Spontaneous NO Releaser. Br J Pharmacol. 1994;113:1137-1140.3. Y. Kita, et al., Antiplatelet Activities of FK409, a New Spontaneous NO Releaser. Br J Pharmacol. 1994;113:385-388.4. Y. Kita, et al., Spontaneous Nitric Oxide Release Accounts for the Potent Pharmacological Actions of FK409. Eur J Pharmacol. 1994;257:123-130.5. M. Hino, et al., FK409, a Novel Vasodilator Isolated from the Acid-treated Fermentation Broth of Streptomyces Griseosporeus I. Taxonomy, Fermentation, Isolation, and Physico-chemical and Biological Characteristics. J Antibiot. 1989; 42:1578-1583.6. J. Decout, et al., Decomposition of FK409, a New Vasodilator: Identification of Nitric Oxide as Metaborite. Bioorg Med Chem Lett. 1995;5:973-978.7. S. Fukuyama, et al., A New Nitric Oxide (NO) Releaser: Spontaneous NO Release from FK409. Free Radic Res. 1995;23:443-452.8. Y. Kita, et al., FR144420, a Novel, Slow, Nitric Oxide-releasing Agent. Eur J Pharmacol. 1995;275:125-130.9. M. Kato, et al., New Reagents for Controlled Release of Nitric Oxide. Structure-stability Relationships. Bioorg Med Chem Lett. 1996;6:33-38.10. Y. Kita, et al., FK409, a Novel Spontaneous NO Releaser: Comparative Pharmacological Studies with ISDN. Cardiovasc Drug Rev. 1996;14:148-165.11. Y. Hirasawa, et al., Antianginal Effects of FR144420, a Novel Nitric Oxide-releasing Agent. Eur J Pharmacol. 1996;303:55-59.12. M. Sato, et al., Nitric Oxide Raises Cytosolic Concentrations of Ca2+ in Cultured Nodose Ganglion Neurons from Rabbits. Neurosci Lett. 1996;206:69-72.13. Y. Kita, et al., Oral Biological Activities of Spontaneous Nitric Oxide Releaser are Accounted for by their Nitric Oxide-releaseing Rates and Oral Absorption Manners. J Pharmacol Exp Ther. 1996;276:421-425.14. S. Fukuyama, et al., Structure-activity Relationships of Spontaneous Nitric Oxide Releasers, FK409 and its Derivatives. J Pharmacol Exp Ther. 1997;282:236-242.15. Y. Kita, et al., Comparison of Hemodynamic Effects of Nitric Oxide (NO) Donors with Different NO-releasing Properties in Rats. J Cardiovasc Pharmacol. 1997;30:223-228.16. Y. Hirasawa, et al., Comparison of Antiplatelet Effect of two Nitric Oxide-donating Agents, FR146801 and FK409. Thromb Haemost. 1998;79:620-624.
How do I prepare a stock solution?

Prepare 10-50 mM NOR solution with DMSO. The DMSO should be dried. Then add enough NOR solution to the cell culture to obtain a suitable concentration of NOR.

What is the solubility of NOR compounds?

NOR 1: 100 mg per 100 ml DMSO (4.3 M)NOR 3: 137 mg per 100 ml DMSO (6.4 M)NOR 4: 30 mg per 100 ml DMSO (1.0 M)NOR 5: 30 mg per 100 ml DMSO (0.9 M)

Is oral administration possible?

Yes. Please review the article by Kita and colleagues (Eur. J. Pharmacol., 257, 123-130, 1994).

How many NO molecules does each NOR molecule release in physiological conditions? What are the byproducts?

On average, each NOR molecule releases from 1 to 1.5 NO molecules in physiological conditions. Unfortunately, the structure of NOR byproducts remains unclear. However, the NOR byproducts have no cytotoxicity at the normal concentration for NO release experiments.

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

 

Dojindo,SIN-1/25/S264,SIN-1 是血管扩张剂 molsidomine 的代谢物

SIN-1 是血管扩张剂 molsidomine 的代谢物，用于分别评估 NO 和过氧亚硝酸盐与其他 NO 供体的有效性。 SIN-1 在分子氧存在下自发分解生成 NO 和超氧化物。 两种产物都非常迅速地结合形成过氧亚硝酸盐（速率常数 k：3.7×10-7 M-1s-1）。 因此，SIN-1 是一种有用的化合物，可以有效地生成过氧亚硝酸盐。 过氧亚硝酸盐是一种非常强的氧化剂，在生理条件下会产生羟基和亚硝基二氧基自由基。 过氧亚硝酸盐也在酸性条件下快速分解生成硝酸根离子，在碱性条件下缓慢分解生成硝酸根离子。 这些物种具有与 NO 不同的生物活性。

Reaction of NO release and peroxynitrite production

1. M. Feelisch, et al., On the Mechanism of NO Release from Sydnonimines. J Cardiovasc Pharmacol. 1989;14:S13-S22.2. M. Feelisch, The Biochemical Pathways of Nitric Oxide Formation from Nitrovasodilators: Appropriate Choice of Exogenous NO Donors and Aspects of Preparation and Handling of Aqueous NO Solutions. J Cardiovasc Pharmacol. 1991;17:S25-S33.3. N. Hogg, et al., Production of Hydroxyl Radicals from the Simultaneous Generation of Superoxide and Nitric Oxide. Biochem J. 1992;281:419-424.4. M. E. Murphy, et al., Nitric Oxide Hyperpolarizes Rabbit Mesenteric Arteries via ATP-sensitive Potassium Channels. J Physiol. 1995;486:47-58.5. H. Kankaanranta, et al., 3-Morpholino-sydnonimine-induced Suppression of Human Neutrophil Degranulation in Not Mediated by Cyclic GMP, Nitric Oxide or Peroxynitrite: Inhibition of the Increase in Intracellular Free Calcium Concentration by N-Morpholinoiminoacetoni. Mol Pharmacol. 1997;51:882-888.6. S. Yamamoto, et al., Subarachnoid Hemorrhage Impairs Cerebral Blood Flow Response to Nitric Oxide but Not to Cyclic GMP in Large Cerebral Arteries. Brain Res. 1997;757:1-9.7. S. Pfeiffer, et al., Interference of carboxy-PTIO with Nitric-oxide and Peroxynitrite-mediated Reactions. Free Radic Biol Med. 1997;22:787-794.8. M. B. Herrero, et al., Tyrosine Nitration in Human Spermatozoa: A Physiological Function of Peroxynitrite, the Reaction Product of Nitric Oxide and Superoxide. Mol Hum Reprod. 2001;7:913-921.9. P. D. Lu, et al., Cytoprotection by Pre-emptive Conditional Phosphorylation of Translation Initiation Factor 2. EMBO J. 2004;23:169-179.

细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。

Dojindo,DTC Na/100/D465

二乙基二硫代氨基甲酸酯 (DETC) 是一种很好的体内一氧化氮自旋捕获试剂。然而，由于其水溶性差，DETC尚未广泛用于生物样品中的NO检测。 DTCS 是 DETC 的类似物，可形成水溶性铁 (II) 络合物 (Fe-DTCS)。然后 Fe-DTCS 配合物与 NO (NO-Fe-DTCS) 形成配合物。 Yoshimura 博士成功获得了脂多糖在小鼠腹膜中诱导的 NO 的二维 ESR 图像。本实验使用 DTCS 钠盐 (DTCS Na)，因为它的毒性低于铵盐（钠盐 LD50：1942 mg/kg；铵盐 LD50：765 mg/kg）。由于 Fe-DTCS 配合物在空气或水溶液中比其他二硫代氨基甲酸盐配合物更稳定，因此它可能是用于生化研究的有用的自旋捕获试剂。 Fe-DTCS 复合物应在制备后立即使用。需要过量的 DTCS Na（通常为 5 当量的 DTCS Na 到 FeSO4）才能形成更稳定的溶液。二硫代氨基甲酸盐在生理条件下倾向于分解形成有毒的二硫化碳。
Reaction of NO quenching

Preparation of Fe(II)-DTCS Complex1. 用 20 ml 水溶解 278 mg FeSO4, 7H2O（七水硫酸亚铁）a) 以制备 50 mM FeSO4 溶液。b)2。用 10 ml watera) 溶解 123 mg DTCS Na，以制备 50 mM DTCS solution.3。将 1 ml DTCS Na 溶液与 8.8 ml 缓冲溶液a)（pH 7 或更高）混合。在使用前加入 200 μl FeSO4 溶液。c)a) 在溶解 FeSO4 之前，通过氮气鼓泡至少 30 分钟清除水中或缓冲液中的任何溶解氧。b) FeSO4 溶液可在 -20ºC 下储存至少 2 个月。c) Fe(II)-DTCS 复合物是无色的。如果溶液呈棕色，则可能由于溶液中溶解氧而形成了 Fe(III)-DTCS。但棕色溶液仍可用于NO捕集。NO-Fe(II)-DTCS配合物的制备1．在氩气流下，将 200 μl FeSO4 溶液添加到 9.4 ml 缓冲溶液（pH 7 或更高）中。然后通过玻璃毛细管将 NO 气体鼓泡 15 分钟，将 NO 引入溶液中。2。向 FeSO4 溶液中加入 400 μl DTCS Na 溶液，继续鼓泡 5 分钟引入 NO。 3．用氩气鼓泡 5 分钟去除多余的 NO，并储存在 -20ºC。 NO-Fe(II)-DTCS 溶液可在 -20ºC 无氧条件下储存至少 2 个月。

1. T. Yoshimura, et al., In vivo EPR Detection and Imaging of Endogenous Nitric Oxide in Lipopolysaccharide-treated Mice. Nat Biotechnol. 1996;14:992-994.2. B. Kalyanaraman, Detectionof Nitric Oxide by Electron Spin Resonance in Chemical, Photochemical, Cellular, Physiological, and Pathophysiological Systems. Methods Enzymol. 1996;268:168-187.3. H. Yokoyama, et al., In vivo ESR-CT Imaging of the Liver in Mice Receiving Subcutenous Injection of Nitric Oxide-Bound Iron Complex. Magn Reson Imaging. 1997;15:249-253.

Dojindo,BABE/10/B437,Bromoacetamidobenzyl-EDTA (BABE) 是一种螯合标记试剂与巯基结合

Product Description of BABEs
Bromoacetamidobenzyl-EDTA (BABE) 是一种螯合标记试剂，可与巯基结合。 BABE 的铁螯合物 (FeBABE) 是一种独特的工具，用于确定蛋白质的三维结构以及蛋白质-蛋白质或蛋白质-DNA 复合物的结合结构。 BABE 通过其巯基将 EDTA 部分添加到蛋白质中。 一旦附着在蛋白质上，FeBABE 就会切割附近的肽或 DNA 链。 切割位点在 FeBABE 结合位点的 12 埃以内。 铁 (II)-螯合物在过氧化氢存在下切割肽或 DNA 链。 裂解反应迅速完成：10 秒到 20 分钟的孵育就足够了。 用凝胶电泳如 SDS-PAGE 分析切割片段的大小。

Labeling Procedure1. Dialyze the protein solution in conjugation buffer (10-20 mM MOPS, 0.2 M NaCl, 2 mM EDTA, 5% glycerol, pH 8.0) at 4ºC overnight.2. After dialysis, adjust the protein concentration to 15-30 mM.3. Add 15 ml of 20 mM FeBABE DMSO solution to 1 ml of the protein solution and incubate it at 37ºC for 1 hour. The final concentration of FeBABE is 0.3 mM (10-20X excess to the protein).4. Dialyze the reaction mixture in protein storage buffer (10-20 mM Tris, 0.1-0.2 M KCl, 10 mM MgCl2, 0.1 mM EDTA, 50% glycerol, pH 7.6) at 4ºC overnight.

1. L. H. DeRiemer, C. F. Meares, D. A. Goodwin and C. I. Diamanti, BLEDTA II: Synthesis of a New Tumer-Visualizing Derivative of Co(III)-bleomycin, J. Labelled Compd. Radiopharm., 1981, 18, 1517.2. T. M. Rana and C. F. Meares, Specific Cleavage of a Protein by an Attached Iron Chelate, J. Am. Chem. Soc., 1990, 112, 2457.3. T. M. Rana and C. F. Meares, Transfer of Oxigen from an artificial protease to peptide carbon during proteolysis, Proc. Natl. Acad. Sci. USA, 1991, 88, 10578.4. D. P. Greiner, R. Miyake, J. K. Moran, A. D. Jones, T. Negishi, A. Ishihama, and C. F. Meares, Synthesis of the Protein Cutting Reagent Iron (S)-1-(p-Bromoacetamidobenzyl) ethylebediaminetetraacetate and Conjuation to cysteine Sie Cahins, Bioconjugate Chem., 1997, 8, 44.5. E. Platis, M. R. Ermacora and R. O. Fox, Oxidative Polypeptide Cleavage Mediated by EDTA-Fe Covalently Linked to Cysteine Residue, Biochemistry, 1993, 32, 12761.6. S. L. Traviglia, S. A. Datwyler, D. Yan, A. Ishihama and C. F. Meares, Targeted Protein Footprinting: Where Different Transcription Factors bind to RNA Polymerase, Biochemistry, 1999, 38, 4259.7. J. B. Ghaim, D. P. Greiner, C. F. Meares and R. B. Gennis, Proximity Mapping the suface of Membrane Protein Using an Artificial Protease: Demonstration That the Quinone-Binding Domain of Subunit I Is near the N-Terminal Region of Subunit II of Cytochrome bd, Biochemistry, 1995, 34, 11311.8. R. Miyake, K. Murakami, J. T. Owens, D. P. Greiner, O. N. Ozoline, A. Ishihama and C. F. Meares, Dimeric Association of Escherichia coli RNA Polymerase alfa subunits, studied by Cleavage of Single-Cysteine alfa Sununits Conjugated to Iron-(S)-1-(p-(Bromoacetamido) benzyl)ethylenediaminetetraacetate, Biochemistry, 1998, 37, 1344.9. J. T. Owens, R. Miyake, K. Murakami, A. J. Chmura, N. Fujita, A. Ishihama and C. F. Meares, Mapping the sigma70 subunits contact sites on Escherichia coli RNA polymerase with a sigma70-conjugated chemical protease, Proc. Natl. Acad. Sci. USA, 1998, 95, 6021.10. J. A. Bown, J. T. Owens, C. F. Meares, N. Fujita, A. Ishihama, S. J. Busby and S. D. Minchin, Organization of open complexes at Escherichia coli promoters. Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase, J. Biol. Chem., 1999, 274, 2263.11. F. Colland, N. Fujita, D. Kotlarz, J. A. Bown, C. F. Meares, A. Ishihama and A. Kolb, Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes, EMBO J., 1999, 18, 4049.12. G. M. Heilek, R. Marusak, C. F. Meares and H. F. Noller, Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4, Proc. Natl. Acad. Sci. USA, 1995, 92, 1113.13. G. M. Heilek and H. F. Noller, Site-directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S5, Science, 1996, 272,

Dojindo,S-亚硝基谷胱甘肽/100/N415

Product Description of S-Nitrosothiols

亚硝基硫醇化合物在特定生理条件下释放 NO 并变成二硫化物。虽然大多数 S-亚硝基硫醇化合物不稳定，但 S-亚硝基谷胱甘肽却异常稳定。此外，S-亚硝基谷胱甘肽是水溶性的。尽管 S-亚硝基硫醇是一种良好的 NO 供体，没有硝酸盐耐受性，但有证据表明 S-亚硝基硫醇本身在鸟苷酸环化酶激活期间具有类似 NO 的活性。亚硝基硫醇的另一个重要反应是 NO 转移到其他硫醇化合物。由于它取决于硫醇的 pKa，因此该转移反应在生理 pH 水平下进行。这些亚硝基硫醇的弛豫效率已使用 Rataprta 环样品进行了比较：SNAP > S – N i t r o s o g l u t a t h i o n e = S – N i t r o s o – N-乙酰半胱氨酸>S-亚硝基辅酶 A>S-亚硝基-L-半胱氨酸。 Kowaluk 博士和其他人报告说，从 SNAP 中自发释放 NO 不能解释体外血管舒张。 NO 从亚硝基硫醇化合物中的自发释放可能不是血管舒张的关键因素。在细胞膜上产生的亚硝基硫醇代谢物可能是松弛的必要元素。

1. A. Gibson, et al., An Investigation of Some S-Nitrosothiols, and of Hydroxy-arginine, on the Mouse Anococcygeus. Br J Pharmacol. 1992;107:715-721.2. M. W. Radomski, et al., S-Nitroso-glutathione Inhibits Platelet Activation in Vitro and in Vivo. Br J Pharmacol. 1992;107:745-749.3. R. M. Clancy, et al., Novel Synthesis of S-Nitrosoglutathione and Degradation by Human Neutrophils. Anal Biochem. 1992;204:365-371.4. J. W. Park, et al., Transnitrosation as a Predominant Mechanism in the Hypotensive Effect of S-Nitrosoglutathione. Biochem Mol Biol Int. 1993;30:885-891.5. D. Barrachina, et al., Nitric Oxide Donors Preferencially Inhibit Neuronally Mediated Rat Gastric Acid Secretion. Eur J Pharmacol. 1994;262:181-183.6. E. A. Konorev, et al., S-Nitrosoglutathione Improves Functional Recovery in the Isolated Rat Heart After Cardioplegic Ischemic Arrest-evidence for a Cardioprotective Effect of Nitric Oxide. J Pharmacol Exp Ther. 1995;274:200-2006.7. S. C. Askew, et al., Catalysis by Cu2+ of Nitric Oxide Release from S-Nitrosothiols (RSNO). J Chem Soc Perkin Trans 2. 1995;741-745.8. D. J. Banett, et al., NO-group Transfer(Transnitrosation) between S-Nitrosothiols and Thiols. Part 2. J Chem Soc Perkin Trans 2. 1995;1279-1282.9. J. G. De Man, et al., Effect of Cu2+ on Relaxations to the Nitrergic Neurotransmitter, NO and S-Nitrosothiols in the Rat Gastric Fundus. Br J Pharmacol. 1996;119:990-996.10. A. P. Dicks, et al., Generation of Nitric Oxide from S-Nitrosothiols Using Protein-bound Cu2+ Sources. Chem Biol. 1996;3:655-659.11. J. A. Cook, et al., Convenient Colorimetric and Fluorometric Assays for S-Nitrosothiols. Anal Biochem. 1996;238:150-158.12. S. X. Liu, et al., Nitric Oxide Donors: Effects of S-Nitrosoglutathione and 4-Phenyl-3-furoxancarbonitrile on Ocular Blood Flow and Retinal Function Recovery. J Ocul Pharmacol Ther. 1997;13:105-114.13. C. Alpert, et al., Detection of S-Nitrosothiols and Other Nitric Oxide Derivatives by Photolysis-chemiluminescence Spectrometry. Anal Biochem. 1997;245:1-7.14. T. Akaike, et al., Nanomolar Quantification and Identification of Various Nitrosothiols by High Performance Liquid Chromatography Coupled with Flow Reactors of Metals and Griess Reagent. J Biochem. 1997;122:459-466.

Dojindo,ABD-F/100/A016,ABD-F 通过巯基反应产生高荧光化合物

ABD-F 具有苯并呋喃部分，可通过与巯基反应产生高荧光化合物。 衍生化合物的激发和发射波长分别为 389 nm 和 513 nm。 ABD-F的反应速度比SBD-F快30倍。 在 50ºC、pH 8 的水性条件下，ABD-F 与硫醇化合物的反应在 5 分钟内完成。但是，ABD-F 在这些条件下不与丙氨酸、脯氨酸或胱氨酸反应。 在 pH 2 时可以观察到其最大荧光强度。在反相 HPLC 分析中，可以单独检测预标记的 ABD-硫醇化合物。 检测限 (S/N=3) 为半胱氨酸每次注射 0.6 pmol，谷胱甘肽每次注射 0.4 pmol，N-乙酰半胱氨酸每次注射 1.9 pmol，半胱胺每次注射 0.5 pmol。

ABD Labeling Protocol1. To prepare sample solution, mix or dissolve a sample with 100 mM borate buffer, pH 8.0 containing 2 mM EDTA.2. Mix 500 μl of the sample solution and 500 μl of 1 mM ABD-F/100 mM borate buffer in a reaction vial.3. Heat the vial at 50ºC for 5 minutes and cool it on an ice bath.4. Add 300 μl of 100 mM HCl aqueous solution to the reaction mixture.5. Use this mixture for HPLC analysis to determine ABD-labeled compounds; excitation: 389 nm, emission: 513 nm.

1. T. Toyo’oka, et al., New Fluorogenic Reagent Having Halogenobenzofurazan Structure for Thiols: 4-(Aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole. Anal Chem. 1984;56:2461-2464.2. T. Toyo’oka, et al., Isolation and Characterization of Cysteine-Containing Regions of Proteins Using 4-(Aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole and High-Performance Liquid Chromatography. Anal Chem. 1985;57:1931-1937.3. T. Toyo’oka, et al., Amino Acid Composition Analysis of Minute Amounts of Cysteine-containing Proteins Using 4-(Aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole and 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole in Combination with HPLC. Biomed Chromatogr. 1986;1:15-20.4. T. Toyo’oka, et al., Simultaneous Determination of Thiols and Disulfides by High-performance Liquid Chromatography with Fluorescence Detection. Anal Chim Acta. 1988;205:29-41.5. Y. Luo, et al., Antichymotrypsin interaction with chymotrypsin. Intermediates on the way to inhibited complex formation. J Biol Chem. 1999;274:17733-17741.

Dojindo,ICG-Sulfo-OSu/1/I254,ICG 是用于确定心输出量

ICG 是用于确定心输出量、肝功能和肝血流量以及用于眼科血管造影的染料之一。 它具有长的激发波长和发射波长，分别约为 780 nm 和 800 nm。 由于其在红外区域附近的长波长和低细胞毒性，ICG 被用于标记抗体以进行体内测定。 然而，与蛋白质结合后的荧光强度非常低，因为在激发后会形成 H-二聚体或能量转移至抗体分子。 Kobayashi 博士和其他人报告说，结合物使用 SDS 和 betamecraptoethanol 通过减少疏水性 p-p 相互作用和 IgG 链的分离显着增加了荧光强度。 他们将经过处理的 ICG 偶联的 daclizumab（人源化单克隆抗体）和人源化抗 HER IgG2 单克隆抗体用于体内检测，以特异性观察肿瘤。
Structural Formula
1. Prepare 6.8 nmol antibody solution with pH 8.5 carbonate buffer or bicine buffer (Good’s buffer).2. Add 6.8-68 nmol of ICG-Sulfo-OSu/DMSO solution to the antibody solution and incubate at room temperature for 30 minutes.3. Purify the reaction mixture with a sephadex G50 column.

Dojindo,SBD-F/100/S013,SBD-F 是一种水溶性试剂

SBD-F 是一种水溶性试剂，可与巯基反应生成高荧光化合物。 HPLC分析硫醇化合物如谷胱甘肽、半胱氨酸、N-乙酰半胱氨酸、CoA和BSA的检测限为每次注射100-500 pmol。衍生化合物的激发和发射分别为385 nm和515 nm .

1. K. Imai, et al., A Novel Fluorogenic Reagent for Thiols: Ammonium 7-Fluorobenzo-2-Oxa-1, 3-Diazole-4-Sulfonate. Anal Biochem. 1983;128:471-473.2. T. Toyo’oka, et al., High-Performance Liquid Chromatography and Fluorometric Detection of Biologically Important Thiols, Derivatized with Ammonium 7-Fluorobenzo-2-Oxa-1, 3-Diazole-4-Sulphonate(SBD-F). J Chromatogr. 1983;282:495-500.3. T. Toyo’oka, et al., Fluorescence Analysis of Thiols with Ammonium 7-Fluorobenzo-2-Oxa-1, 3-Diazole-4-Sulphonate. Analyst. 1984;109:1003-1007.4. T. Toyo’oka, et al., Determinaiton of Total Captopril in Dog Plasma by HPLC After Prelabelling with Ammonium 7-Fluorobenzo-2-Oxa-1, 3-Diazole-4-Sulphonate(SBD-F). J Pharm Biomed Anal. 1984;2:473-479.5. T. Sueyoshi, et al., Application of a Fluorogenic Reagent, Ammonium 7-Fluorobenzo-2-Oxa-1, 3-Diazole-4-Sulphonate for Detection of Cystine-Containing Peptides. J Biochem. 1985;97:1811-1813.6. T. Toyo’oka, et al., Simultaneous Determination of Thiols and Disulfides by High-performance Liquid Chromatography with Fluorescence Detection. Anal Chim Acta. 1988;205:29-41.7. T. Araki, et al., The Position of the Disulfide Bonds in Human Plasmaα2HS-glycoprotein and the Repeating Double Disulfide Bonds in the Domain Structure. Biochim Biophys Acta. 1989;994:195-199.8. B. A. McMullen, et al., Location of the Disulfide Bonds in Human Plasma Prekallikrein; The Presence of Four Novel Apple Domains in the Amino-Terminal Portion of the Molecule. Biochemistry. 1991;30:2050-2056.9. D. Hess, et al., Identification of the Disulfide Bonds of Human Complement C1s. Biochemistry. 1991;30:2827-2833.

Dojindo,HiLyte Fluor 647标记试剂盒-NH2/3/LK15

HiLyte Fluor* 647 Labeling Kit-NH2 主要用于制备红色荧光标记的蛋白质，例如用于免疫染色的 IgG，以及用于示踪的细胞蛋白质。 NH2 反应性 HiLyteFluor 647 是该试剂盒的一个组成部分，具有与蛋白质或其他分子上的氨基反应的琥珀酰亚胺基 (NHS)（图 1）。 该试剂盒包含标记所需的所有试剂。 每管 HiLyte Fluor 647 最多可标记 200 μg 的 IgG，每个 IgG 分子可结合约 4 至 6 个 HiLyte Fluor 647 分子。 标记过程很简单——将 NH2 反应性 HiLyte Fluor 647 添加到膜上的 IgG 溶液中，并在 37ºC 下孵育 10 分钟。 多余的 HiLyte Fluor 647 分子可以通过过滤管去除。 HiLyte Fluor 647 标记的 IgG 的激发和发射波长分别为 652 nm 和 673 nm（图 2）。 *HiLyte Fluor 是 AnaSpec, Inc. 的商标。

Fig. 1 Fluorescence Spectrum of HiLyte Fluor 647-conjugated IgG

Fig. 2 Fluorescence spectrum of HiLyte Fluor 647-conjugated IgG______excitation spectrum______emission spectrum

Precaution♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.♦ IgG or HiLyte Fluor 647-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with molecular weights larger than 10,000, such as BSA or gelatin, purify the IgG solution before labeling HiLyte Fluor 647 with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

1. S. Hiroyasu, T. Ozawa, H. Kobayashi, M. Ishii, Y. Aoyama, Y. Kitajima, T. Hashimoto, J.C.R. Jones and D. Tsuruta, “Bullous pemphigoid IgG induces BP180 internalization via a macropinocytic pathway”, Am. J. Pathol.., 2013, 182, (3), 828.2. W. Jin, K. Yamada, M. Ikami, N. Kaji, M. Tokeshi, Y. Atsumi, M. Mizutani, A. Murai, A. Okamoto, T. Namikawa, Y. Baba, M. Ohta, “Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products”, J. Microbiol. Methods., 2013, 92, (3), 323.3. Y. Hayashi, M. Okutani, S. Ogawa, T. Tsukahara, R. Inoue, “Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis”, Anim. Sci. J.., 2018, 89, (5), 825.

Can I use this kit for other proteins?

是的，如果分子量大于 50,000。

在标记蛋白质之前是否必须使用过滤管？
如果蛋白质溶液不含带氨基的小分子，蛋白质浓度为10 mg/ml，或约70 μM，则无需使用过滤管。 将 10 μl 样品溶液与 90 μl 反应缓冲液混合，然后将 8 μl NH2 反应性 HiLyte Fluor 647（在步骤 3 中制备）添加到混合物中，然后按照从步骤 4 开始的方案进行操作。

How long is the HiLyte Fluor 555-labeled protein stable?

If you store at 4oC, it is stable for over 2 months. For longer storage, add 100% volume of glycerol, aliquot, and store at -20oC. However, please note that stability depends on the protein itself.

What is the minimum amount of IgG that can be labeled by this kit?

The minumum amount of IgG is 10 μg. For less than 20 μg, follow the manual and add 4 μl NH2-reactive HiLyte Fluor 647 instead of 8 μl at step 4.

Can I use this kit to label oligonucleotides or peptides?

No. Oligonucleotides and peptides may be too small to retain on the membrane filter of the Filtration tube.

Dojindo,DPPP/10/D350,DPPP 是一种非荧光三苯基膦化合物

DPPP 是一种非荧光三苯基膦化合物。 它与氢过氧化物反应生成 DPPP 氧化物，在 352 nm 激发和 380 nm 发射波长下发射荧光（图 1）。 柱后 HPLC 法用于测定样品溶液中的过氧化磷脂。
Fig 1. DPPP reaction with lipid peroxidase
1. K. Akasaka, et al., Study on Aromatic Phosphines for Novel Fluorometry of Hydroperoxides(II) – the Determination of Lipid Hydroperoxides with Diphenyl-1-Pyrenylphosphine-. Anal Lett. 1987;20:797-807.2. K. Akasaka, et al., An Aromatic Phosphine Reagent for the HPLC-fluorescence Determination of Hydroperoxides -Determination of Phosphatidylcholine Hydroperoxides in Human Plasma. Anal Lett. 1988;21:965-975.3. K. Akasaka, et al., A Simple Fluorometry of Hydroperoxides in Oils and Foods. Biosci Biotech Biochem. 1992;56:605-607.4. K. Akasaka, et al., High-performance Liquid Chromatography and Post-Column Derivatization with Diphenyl-1-Pyrenylphosphine for Fluorimetric Determination of Triacylglycerol Hydroperoxides. J Chromatogr. 1992;596:197-202.5. K. Akasaka, et al., Simultaneous Determination of Hydroperoxides of Phosphatidylcholine, Cholesterol Esters and Triacylglycerols by Column-Switching High-Performance Liquid Chromatography with a Post-column Detection system. J Chromatogr. 1993;622:153-159.6. K. Akasaka, et al., Normal-phase High-performance Liquid Chromatograohy with a Fluorimetric Postcolumn Detection System for Lipid Hydroperoxides. J Chromatogr A. 1993;628:31-35.7. Y. Okimoto, et al., A Novel Fluoresceint Probe Diphenyl-pyrenylphosphine to Follow Lipid Peroxidation in Cell Membranes. FEBS Lett. 2000;474:137-140

Dojindo,用于线粒体单线态氧成像的 Si-DMA/2/MT05

单线态氧 (1O2) 是活性氧 (ROS) 中的一种。众所周知，1O2 是导致皮肤出现斑点和皱纹的原因，因为它具有很强的氧化潜力。在癌症研究领域，1O2 特别重要，因为它在光动力疗法 (PDT) 中发挥着关键作用，这是一种使用光辐照和光敏剂的新兴抗癌疗法。对于1O2的检测，现有的单线态氧荧光检测试剂由于其细胞膜不透性，不能用于活细胞。
马岛等人。合成了一种由含硅罗丹明和蒽部分组成的新型远红荧光探针，即 Si-DMA，分别作为生色团和 1O2 反应位点。在 1O2 存在下，由于蒽部分 (1),(2) 形成内过氧化物，Si-DMA 的荧光增加了 17 倍。在七种不同的 ROS 中，Si-DMA 能够选择性地检测 1O2（图 3）。此外，Si-DMA 能够用 5-氨基乙酰丙酸 (5-ALA)（血红素的前体）实时显示线粒体中原卟啉 IX 生成 1O2。
1. S. Kim, T. Tachikawa, M. Fujitsuka, T. Majima, “Far-Red Fluorescence Probe for Monitoring Singlet Oxygen during Photodyanamic Therapy”, J. Am. Chem. Soc., 2014, 136 (33), 11707-11715.

Usage ExampleFluorescence microscopic detection of 1O2 in HeLa cells after added 5-aminolevulinic acid (5-ALA)1. HeLa cells (2.4×105 cells/ml, Hanks’ HEPES buffer 200 μl) were seeded on a μ-slide 8 well (Ibidi) and cultured at 37oC in a 5%CO2 incubator overnight.2. Cells were washed with 200 μl Hanks’ HEPES buffer twice.3. 150 μg/ml of 5-ALA in Hanks’ HEPES buffer was added to the μ-slide and cultured at 37oC in a 5%CO2 incubator for 4 hours.4. Cells were washed with 200 μl Hanks’ HEPES buffer twice.5. 200 μl of Si-DMA working solution (40 nmol/L) was added and cultured at 37oC in a 5%CO2 incubator for 45 minutes.6. Cells were washed with 200 μl Hanks’ HEPES buffer twice.7. 200 μl Hanks’ HEPES buffer was added and observe the cells under a fluorescence microscope.

Dojindo,2,3-二氨基萘（不检测）/10/D418,Griess 法是检测 NO 浓度的方法

Griess 法是一种简单而流行的检测 NO 浓度的方法。 2,3-二氨基萘 (DAN) 是 Griess 测定法的高灵敏度替代方法。 DAN 方法的灵敏度比 Griess 检测高 50-100 倍：Griess 检测的检测限为 1 mM，而 DAN 方法的检测限为 10-50 nM。 DAN 在酸性条件下与 NO2– 反应生成荧光萘三唑。萘三唑的最大发射波长为410 nm。但是，建议在 450 nm 处检测以避免荧光空白并提高灵敏度。 DAN 的荧光背景很低，可实现最大灵敏度。确定了 DAN 与 NO2- 的最佳反应条件。反应应在 pH 2 和室温下进行 5 分钟，萘三唑产生的荧光应在 pH 10 或更高时测定。 DAN 是一种光敏试剂，有时会变成深棕色晶体。由于这种棕色产物不能用于荧光检测，因此需要重结晶。

Reaction of 2,3-Diaminonaphthalene with NO2

NO2 Assay Using 2,3-Diaminonaphthalene (DAN)1. Dissolve 50 μg DAN in 1 ml 0.62 M HCl to prepare 0.31 mM DAN solution.a)2. Mix 10 μl DAN solution with 100 μl NaNO2 solution (0-10 mM) or sample solution. Incubate the mixture at room temperature for 10-15 minutes.3. Add 5 μl 2.8 M NaOH solution to the reaction solution.b)4. Dilute 100 μl of this solution with 4 ml water, followed by fluorescent measurement with excitation wavelength at 365 nm and emission wavelength at 450 nm.5. Prepare a calibration curve using this data where the X-axis is NaNO2 concentration and the Y-axis is fluorescence intensity. Then, use this calibration curve to determine the NO2 concentration of the sample solution.

a) Acidic conditions are required for a rapid reaction.b) Basic conditions (pH 10 or higher) are required for a high fluorescence signal.

C. R. Sawicki, Anal. Lett., 4, 761 (1971);P. Damiani, et al., Talanta, 8, 649 (1986);W. R. Tracey, et al., J. Pharmacol. Exp. Ther., 252, 922 (1990);J. S. Pollock, et al., Proc. Natl. Acad. Sci. USA, 88, 10480 (1991);T. P. Misko, et al., Anal. Biochem., 214, 11 (1993);R. G. Tilton, et al., Invest. Ophthamol. Vis. Sci., 35, 3278 (1994);T. A. Mayer, et al., J. Surg. Res., 58, 570 (1995);Y. Kono, et al., Biochem. J., 312, 947 (1995);R. Metheringham, et al., Microbiology, 143, 2647 (1997);A. M. Rao, et al., Brain Res., 793, 265 (1998);N. Nakatsubo, et al., Biol. Pharm. Bull., 21, 1247 (1998);D. Jourd’heuil, et al., Arch. Biochem. Biophys., 365, 92 (1999).

Dojindo,Sulfo-KMUS/50/S250

杂双功能交联剂具有活化的酯和马来酰亚胺反应基团。 这些官能团分别与蛋白质的胺和巯基反应。 酶标记的半抗原是使用异双功能交联剂（例如 EMCS 或 GMBS）制备的。 交联反应需要中性 pH 值和温和的温度，因为在交联反应中需要保持酶活性和抗体滴度。 可使用具有 3、5、7 或 10 个线性碳链的异双功能交联剂。 这些线性脂肪链充当两个反应位点与其水溶性试剂之间的间隔物。 在更宽的 pH 范围内，它们比芳香族交联剂（例如 succinimidyl-4-N-maleimidobenzoate）更稳定。

Conjugation of Macromolecules with Hetero-Bifunctional Cross-Linking Reagent

Hetero-bifunctional Reagents

Product Name Code Length (Å)
Sulfo-EMCS S024 9.4
Sulfo-GMBS S025 6.9
Sulfo-HMCS S026 13.0
Sulfo-KMUS S250 16.7
Sulfo-SMCC S330 8.0
1. J. V. Staros, N-Hydroxysulfosuccinimide Active Esters:Bis(N-hydroxysulfosuccinimide) Esters of Two Dicarboxylic acids Are Hydrophilic, Membraneimpermeant, Protein Cross-linkers, Biochemistry, 1982, 21, 3950.2. P. S. R. Anjaneyulu, James V. Staros, Reactions of N-Hydroxysulfosuccinimide Active Esters, Int. J. Peptide Protein Res., 1987, 30, 117.3. Y. Fukami, K. Sato, K. Ikeda, K. Kamisango, K. Koizumi and T. Matsuno, Evidence for Autoinhibitory Regulation of the c-src Gene Product a Possible Interaction Between the Src Homology 2 Domain and Autophosphorylation Site, J. Biol. Chem., 1993, 268, 1132.

Dojindo,总谷胱甘肽定量试剂盒/100/T419

谷胱甘肽 (GSH) 是动物组织、植物组织、细菌和酵母中含量最丰富的硫醇化合物。 GSH 具有许多不同的作用，包括防止活性氧和维持蛋白质硫醇基团。在这些过程中，GSH 被转化为其氧化形式，谷胱甘肽二硫化物 (GSSG)。由于 GSSG 随后被谷胱甘肽还原酶酶促还原，因此 GSH 是生物体中的主要形式。 DTNB（5,5 EDithiobis(2-nitrobenzoic acid)）被称为 Ellman 试剂，用于检测硫醇化合物。 1985年，M. E. Anderson博士提出DTNB和谷胱甘肽还原酶的谷胱甘肽循环系统可以作为一种高灵敏度的谷胱甘肽检测方法。 DTNB 和 GSH 反应生成图 1 中的 5-Mercapto-2-nitrobenzoic acid (TNB)。由于 TNB 是黄色的，样品溶液中的 GSH 浓度可以通过 O.D. 确定。在 412 nm 吸光度下测量（图 2）。 GSH 通过谷胱甘肽还原酶从 GS-TNB 再生，并再次与 DTNB 反应生成 TNB。这种再循环反应提高了总谷胱甘肽检测的灵敏度。总谷胱甘肽定量试剂盒包含总谷胱甘肽测量所需的所有试剂，样品制备中使用的试剂除外。推荐使用 5-磺基水杨酸从样品溶液中去除蛋白质，并防止 GSH 氧化和 γ-谷氨酰转肽酶反应。但是，样品制备的最佳方法因样品而异，因此请查看参考资料。该试剂盒可用于使用标准方法定量 1 μM 至 100 μM 的总谷胱甘肽浓度。对于较低的谷胱甘肽浓度，例如在血液样本中，需要更长的孵育时间。
Mechanism of total glutathione quantificationAbsorption spectrum of 5-Mercapto-2-nitrobenzoic acid in phosphate buffer (pH 7.5)

Assay Procedure

Preparation of Various Sample SolutionCells (Adhesive cells: 5×105 cells; Leukocyte cells: 1×106 cells)1. Collect cells by centrifugation at 200 g for 10 min at 4°C. Discard the supernatant.2. Wash the cells with 300 μl PBS and centrifuge at 200 g for 10 min at 4°C. Discard the supernatant.3. Add 80 μl 10 mM HCl, and lyse the cells by freezing and thawing twice.4. Add 20 μl 5% SSA and centrifuge at 8,000 g for 10 min.5. Transfer the supernatant to a new tube, and use it for the assay. If the final concentration of SSA is over 1%, add ddH2O to reduce the concentration of SSA from 0.5 to 1%.Tissue (100 mg)1. Homogenize the tissue in 0.5-1.0 ml 5% SSA.2. Centrifuge the homogenized tissue sample at 8,000 g for 10 min.3. Transfer the supernatant to a new tube and add ddH2O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay.

Plasma1. Centrifuge anticoagulant-treated blood at 1,000 g for 10 min at 4°C.2. Transfer the top plasma layer to a new tube and add 5% SSA equivalent to half of the volume of the plasma.3. Centrifuge at 8,000 g for 10 min at 4°C4. Transfer the supernatant to a new tube, and add ddH2O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay.

Erythrocytes1. Centrifuge anticoagulant-treated blood at 1,000 g for 10 min at 4°C.2. Discard the supernatant and the white buffy layer.3. Lyse the erythrocytes with 5% SSA equivalent to 4 times the volume of the erythrocytes.4. Centrifuge at 8,000 g for 10 min at 4°C.5. Transfer the supernatant to a new tube, and add ddH2O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay. Erythrocytes can be isolated from the remaining sample solution after the plasma sample isolation.

Preparation of Assay SolutionsPreparation of 5% 5-Sulfosalicylic Acid (SSA) SolutionNote: SSA is not included in this kit.1. Dissolve 1 g SSA in 19 ml water.2. Store the solution at 4°C (stable for 6 months at 4°C).

Substrate Working SolutionAdd 1 ml Buffer Solution to 1 vial of Substrate, and dissolve. Substrate working solution is stable for 2 months at -20ºC.Enzyme Working SolutionMix Enzyme solution with pipetting before using. Take out 20 μl Enzyme solution and mix it with 4 ml Buffer solution. Enzyme working solution is stable for 2 months at 4ºC.Coenzyme Working SolutionAdd 0.7 ml ddH2O to the Coenzyme vial and dissolve. If you don’t use all of the coenzyme working solution in one day, aliquot it into microtubes and store at -20ºC. If you use all of the coenzyme working solution in one day, just add 6.3 ml Buffer solution to the vial.The Coenzyme vial is under vacuum pressure; carefully open the cap or use a syringe to add Buffer solution. Since the Coenzyme working solution dissolved in the Buffer solution is not stable, use it in one day. The coenzyme solution prepared with ddH2O is only stable for 2 months at -20oC. Dilute 10 times with Buffer solution to prepare Working solution prior to use.GSH Standard SolutionsTo prepare 200 μM GSH standard solution, add 2 ml of 0.5-1% SSA to the Standard GSH vial and dissolve. Dilute 100 μl of the 200 μM GSH standard solution with 100 μl of 0.5% SSA, and repeat using serial dilution to prepare the following GSH standard solutions:100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM and 0.The Standard GSH vial is under vacuum pressure; carefully open the cap or use a syringe to add SSA. GSH powder is difficult to see. The GSH standard solutions are stable for 2 months at -20°C.

Total Glutathione Detection – Standard MethodDetection Range: 5-100 μM1. To each well, add 20 μl of Enzyme working solution, 140 μl of Coenzyme working solution, and 20 μl of either one of the GSH standard solutions or the sample solution.a)2. Incubate the plate at 37°C for 10 min.3. Add 20 μl of Substrate working solution, and incubate the plate at 37°C for 5-10 min.4. Read the absorbance at 405 nm or 415 nm using a microplate reader.5. Determine the concentration of GSH in the sample solution using a calibration curveb).

a) Adjust the concentration of SSA in the sample solution to 0.5-1% with ddH2O before the assay. High concentrations of SSA (>1 %) interfere with the assay.b) Since the colorimetric reaction is stable and the O.D. increases linearly over 30 min, GSH concentration can be determined by kinetic or pseudo-endpoint (no stopping reaction, quick measurement of O.D. at certain time periods between 5 and 10 min) methods.

Total Glutathione Detection – High Sensitivity Method Detection Range: 0.5-25 μM1. To each well, add 20 μl of Enzyme working solution, 140 μl of Coenzyme working solution, and 20 μl of either one of the GSH standard solutionsa) or the sample solutionb).2. Incubate the plate at 30°C for 10 min.3. Add 20 μl of Substrate working solution, and incubate the plate at 37°C for 20-40 min.4. Read the absorbance at 405 nm or 415 nm using a microplate reader.5. Determine the concentration of GSH in the sample solution using a calibration curve.

a) Prepare 50 mM GSH standard solution, and then prepare different concentrations of GSH standard solutions by serial dilution with 0.5% SSA as follows: 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, 0.39 μM and 0.b) Adjust the concentration of SSA in the sample solution to 0.5-1% with ddH2O before the assay. Higher concentrations of SSA (>1%) interfere with the assay.

Determination of Total Glutathione (GSH and GSSG) ConcentrationDetermine the total glutathione concentration in the sample solution using the following equations. Since the values obtained by these equations are the amount of total glutathione in treated sample solutions, further calculations are necessary if the actual concentration of glutathione in cells or tissues needs to be determined.

1. G. L. Ellman, Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70-77.2. O. W. Griffith, Determination of Glutathione and Glutathione Disulfide Using Glutathione Reductase and 2-Vinylpyridine. Anal Biochem. 1980;106:207-212.3. M. E. Anderson, Determination of Glutathione and Glutathione Disulfide in Biological Samples. Methods Enzymol. 1985;113:548-555.4. M. A. Baker, et al., Microtiter Plate Assay for the Measurement of Glutathione and Glutathione Disulfide in Large Numbers of Biological Samples. Anal Biochem. 1990;190:360-365.5. C. Vandeputte, et al., A Micrototer Plate Assay for Total Glutathione and Glutathione Disulfide Contents in Cultured/isolated Cells: Performance Study of a New Miniaturized Protocol. Cell Biol Toxicol. 1994;10:415-421.6. S. A. McGrath-Morrow, et al., Inhibition of Glutamine Synthetase in A549 Cells During Hyperoxia. Am J Respir Cell Mol Biol. 2002;27:99-106.7. T. Sato, et al., Senescence Marker Protein-30 Protects Mice Lungs from Oxidative Stress, Aging, and Smoking. Am J Respir Crit Care Med. 2006;174:530-537.8. M. L. Mulhern, et al., The Unfolded Protein Response in Lens Epithelial Cells from Galactosemic Rat Lenses. Invest Ophthalmol Vis Sci. 2006;47:3951-3959.

Calibration Curve

Fig. 4 Calibration curves prepared using pseudo-endpoint method and kinetic methodA) Calibration curve prepared using pseudo-endpoint method. 10 min incubation at room temperature.B) Calibration curve prepared using kinetic method.

Pseudo-endpoint method:Total glutathione = (O.D.sample O.D.blank) / slopea)Kinetic method:Total glutathione = (Slopesampleb)-Slopeblankb))/slopeb)

a)The slope of the calibration curve prepared by the pseudo-endpoint or kinetic method.b)The slope of the kinetic reaction.

Do I have to dilute the sample solution prior to the assay?
If you do not know the total glutathione level of your sample, multiple dilutions may be necessary. If the total glutathione level of your sample is less than 100 μM, no dilution is necessary.
What interferes with the assay?
Reducing agents (such as ascorbic acid, beta-mercaptoethanol, dithiothreitol, and cysteine) and thiol reactive compounds (such as maleimides) interfere with the glutathione assay. Therefore, reducing agents and thiol reactive compounds should be avoided during the sample preparation.

Dojindo,过氧化物酶标记试剂盒-NH2/1/LK11,Peroxidase Labeling Kit-NH2

过氧化物酶标记试剂盒-NH2 主要用于制备过氧化物酶标记的 IgG 用于酶免疫分析 (EIA) 和制备过氧化物酶标记的抗原用于竞争性 EIA。 NH2 反应性过氧化物酶是该试剂盒的一个组成部分，具有琥珀酰亚胺基 (NHS)，可与结构中具有氨基的蛋白质或其他分子发生反应（图 1）。该试剂盒包含标记过程所需的所有试剂，包括储存缓冲液。标记过程很简单：将 IgG 与 NH2 反应性过氧化物酶混合并在 37ºC 下孵育 2 小时。 NH2 反应性过氧化物酶无需任何激活过程即可与靶分子形成共价键。 NHS 与过氧化物酶的距离约为 1.2 nm，是过氧化物酶分子半径的一半。因此，当过氧化物酶标记的IgG用于EIA时，NH2反应性过氧化物酶的标记效率足够高，可以省去标记后的纯化过程。此外，过氧化物酶标记不会影响目标分子的亲和力。如果标记后需要高纯度偶联物，只需使用亲和柱或凝胶渗透柱即可。标记小分子时，可以使用该套件中包含的过滤管去除多余的分子。因为 NH2 反应性过氧化物酶的氨基被封闭，所以不可能进行自结合。

Fig.1 IgG labeling reaction of NH2-reactive peroxidase

Precaution♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.♦ The molecular weight of the small amine compound to be labeled with this kit should be smaller than 5,000.♦ IgG or peroxidase-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with a molecular weight greater than 10,000, such as BSA or gelatin, purify the IgG solution before labeling peroxidase with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).♦ If the IgG solution contains small insoluble materials,centrifuge the solution and use the supernatant for labeling.

1. A. Miyagawa-Yamaguchi, N. Kotani, and K. Honke, “Expressed Glycosylphosphatidylinositol-Anchored Horseradish Peroxidase Identifies Co-Clustering Molecules in Individual Lipid Raft Domains”, PLoS ONE., 2014, 9, (3), e93054.2. J. Zhang, D. Klufas, K. Manalo, K. Adjepong, J.O. Davidson, G. Wassink, L. Bennet, A.J. Gunn, E.G. Stopa, K. Liu, M. Nishibori, and B.S. Stonestreet, “HMGB1 Translocation After Ischemia in the Ovine Fetal Brain”, J. Neuropathol. Exp. Neurol.., 2016, 75, (6), 527.3. M. Okumura, T. Ozawa, H. Hamana, Y. Norimatsu, R. Tsuda, E. Kobayashi, K. Shinoda, H. Taki, K. Tobe, J. Imura, E. Sugiyama, H. Kishi, and A. Muraguchi, “Autoantibodies reactive to PEP08 are clinically related with morbidity and severity of interstitial lung disease in connective tissue diseases”, Eur. J. Immunol.., 2018, 48, (10), 1717.4. R. Sugisawa, G. Komatsu, E. Hiramoto, N. Takeda, K. Yamamura, S. Arai, and T. Miyazaki, “Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice”, Sci. Rep.., 2018, 8, 13157.5. S. Takatsuka, T. Inukai, S. Kawakubo, T. Umeyama, M. Abe, K. Ueno, Y. Hoshino, Y. Kinjo, Y. Miyazaki, and S. Yamagoe, “Identification of a Novel Variant Form of Aspergillus fumigatus CalC and Generation of Anti-CalC Monoclonal Antibodies”, Med Mycol J., 2019, 60, (1), 11.6. T. Sasaki, K. Liu,T. Agari, T. Yasuhara, J. Morimoto, M. Okazaki, H. Takeuchi, A. Toyoshima, S. Sasada, A. Shinko, A. Kondo, M. Kameda, I. Miyazaki, M. Asanuma, CV. Borlongan, M. Nishibori, and I. Date, “Anti-high mobility group box 1 antibody exerts neuroprotection in a rat model of Parkinson’s disease”, Exp. Neurol.., 2016, 275, 220.7. T. Tsumuraya, I. Fujii, M. Inoue, A. Tatami, K. Miyazaki, and M. Hirama, “Production of monoclonal antibodies for sandwich immunoassay detection of ciguatoxin 51-hydroxyCTX3C”, Toxicon., 2006, 48, (3), 287.8. W. Jin, K. Yamada, M. Ikami, N. Kaji, M. Tokeshi, Y. Atsumi, M. Mizutani, A. Murai, A. Okamoto, T. Namikaw, Y. Baba, and M. Ohta, “Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products”, J. Microbiol. Methods., 2013, 92, (3), 323.9. W.W.P.N. Weerakoon, M. Sakase, N. Kawate, M.A. Hannan, N. Kohama, and H. Tamada, “Plasma IGF-I, INSL3, testosterone, inhibin concentrations and scrotal circumferences surrounding puberty in Japanese Black beef bulls with normal and abnormal semen”, Theriogenology., 2018, 114, (1), 54.10. Y. Watanabe, Y. Kazuki, K. Kazuki, M. Ebiki, M. Nakanishi, K. Nakamura, M. Yoshida Yamakawa,H. Hosokawa, T. Ohbayashi, M. Oshimura, and K. Nakashima, “Use of a Human Artificial Chromosome for Delivering Trophic Factors in a Rodent Model of Amyotrophic Lateral Sclerosis”, Mol Ther Nucleic Acids., 2015, 4, (10), e253.11. Y.S. Kim, D.H. Jung, I.S. Lee, B.J. Pyun and J.S. Kim, “Osteomeles schwerinae extracts inhibits the binding to receptors of advanced glycation end products and TGF-β1 expression in mesangial cells under diabetic conditions”, Phytomedicine., 2016, 23, (4), 388.

Sandwich ELISA

Fig. 2 Sandwich ELISA of CAT (chloramphenicol acetyl transferase) assay.

Plate: 2 μg/ml anti-CAT antibody (rabbit anti sera)-coated high binding plateCAT: 0-400 x 10-3units/ml PBSTPeroxidase-conjugated anti-CAT antibody: Prepared by Peroxidase Labeling Kit-NH2.1μg/ml PBST+blocking reagentSubstrate: TMB peroxidase substrate

Western blot

Fig. 3 Western blot using peroxidase-labeled monoclonal antibody to SIV p24 Gag(2F12).

SIV P55 and molecular weight markers were analyzed in blot 1, 2, and 3.Blot 1: conjugate prepared using Peroxidase Labeling Kit-NH2Blot 2: conjugate prepared using Peroxidase Labeling Kit-SHBlot 3: primary antibody and peroxidase-conjugated secondary antibody (commercially available).

The western blotting using peroxidase-labeled primary antibody gives abetter result than using peroxidase-labeled secondary antibody. In most cases, the sensitivity of the conjugate prepared with Peroxidase/ Alkaline phosphatase Labeling Kit-SH is higher than Labeling Kit-NH2 due to the site specific conjugation on the antibody.

Can I use this kit for Fab or Fab Elabeling?

Yes, you can label Fab or Fab Eusing this kit. The recovery of the conjugate should be over 80%.

Can I use this kit for other proteins?

Yes, if the molecular weight is greater than 50,000 or less than 5,000 and it has a reactive primary or secondary amino group. If the molecular weight is higher than 50,000, follow the labeling protocol for IgG and use 0.5-1 nmol of sample protein for LK11-10.If the molecular weight is less than 5,000, follow the labeling protocol for small molecules. If the molecular weight is higher than 5,000 but lower than 50,000, contact our customer service at info@dojindo.com or 1-877-987-2667 for more information.

Can I use this kit to label an oligonucleotide or oligopeptide?

Yes, if the molecular weight is less than 5,000 and it has a reactive primary or secondary amino group. Follow the labeling protocol for small molecules.

What is the minimum amount of IgG that can be labeled with LK11-10?

The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG.Though 10 μg IgG can still be labeled using this kit, the background will be higher.

How many peroxidase molecules per IgG are introduced?

The average number of peroxidase molecule per IgG is 1 to 3.

Does unconjugated NH2-reactive peroxidase still have an activated ester after the labeling reaction to IgG?

No. It is completely hydrolyzed during the reaction.

Does NH2-reactive peroxidase form an oligomer during the labeling reaction?

No. Since all amino groups of NH2-reactive peroxidase are blocked, no oligomerization is possible.

Do I have to use Storage buffer included with the kit?

No, you do not have to use Storage buffer from the kit. You can choose any kind of buffer appropriate for your experiment.However, the Storage buffer helps to increase the stability of the peroxidase conjugate.

Does Storage buffer contain animal products or polymers?

No, Storage buffer does not contain any animal products, polymers, or heavy metal ions.

Dojindo,Sulfo-HMCS/50/S026,杂双功能交联剂具有活化的酯和马来酰亚胺反应基团

杂双功能交联剂具有活化的酯和马来酰亚胺反应基团。 这些官能团分别与蛋白质的胺和巯基反应。 酶标记的半抗原是使用异双功能交联剂（例如 EMCS 或 GMBS）制备的。 交联反应需要中性 pH 值和温和的温度，因为在交联反应中需要保持酶活性和抗体滴度。 可使用具有 3、5、7 或 10 个线性碳链的异双功能交联剂。 这些线性脂肪链充当两个反应位点与其水溶性试剂之间的间隔物。 在更宽的 pH 范围内，它们比芳香族交联剂（例如 succinimidyl-4-N-maleimidobenzoate）更稳定。

Conjugation of Macromolecules with Hetero-Bifunctional Cross-Linking Reagent

Hetero-bifunctional Reagents

Product Name Code Length (Å)
Sulfo-EMCS S024 9.4
Sulfo-GMBS S025 6.9
Sulfo-HMCS S026 13.0
Sulfo-KMUS S250 16.7
Sulfo-SMCC S330 8.0
1. J. V. Staros, N-Hydroxysulfosuccinimide Active Esters:Bis(N-hydroxysulfosuccinimide) Esters of Two Dicarboxylic acids Are Hydrophilic, Membraneimpermeant, Protein Cross-linkers, Biochemistry, 1982, 21, 3950.2. P. S. R. Anjaneyulu, James V. Staros, Reactions of N-Hydroxysulfosuccinimide Active Esters, Int. J. Peptide Protein Res., 1987, 30, 117.3. Y. Fukami, K. Sato, K. Ikeda, K. Kamisango, K. Koizumi and T. Matsuno, Evidence for Autoinhibitory Regulation of the c-src Gene Product a Possible Interaction Between the Src Homology 2 Domain and Autophosphorylation Site, J. Biol. Chem., 1993, 268, 1132. 

Dojindo,EMCS/100/E018,杂双功能交联剂具有活化的酯和马来酰亚胺反应基团

杂双功能交联剂具有活化的酯和马来酰亚胺反应基团。 这些官能团分别与蛋白质的胺和巯基反应。 酶标记的半抗原是使用异双功能交联剂（例如 EMCS 或 GMBS）制备的。 交联反应需要中性 pH 值和温和的温度，因为在交联反应中需要保持酶活性和抗体滴度。 可使用具有 3、5、7 或 10 个线性碳链的异双功能交联剂。 这些线性脂肪链充当两个反应位点与其水溶性试剂之间的间隔物。 在更宽的 pH 范围内，它们比芳香族交联剂（例如 succinimidyl-4-N-maleimidobenzoate）更稳定。

Conjugation of Macromolecules with Hetero-Bifunctional Cross-Linking Reagent

Hetero-bifunctional Reagents

Product Name Code Length (Å) Solvent
EMCS E018 9.4 DMSO
GMBS G005 6.9 Chloroform, DMF
HMCS H257 13.0 Acetonitrile
KMUS K214 16.7 Acetonitrile, Methyl alcohol

 

1. S. Yoshitake, Y. Yamada, E. Ishikawa and R. Masseyeff, Conjugation of Glucose Oxidase from Aspergillus Niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide, Eur. J. Biochem., 1979, 101, 395.2. T. Kitagawa, T. Shimozono, T. Aikawa, T. Yoshida and H. Nishimura, Preparation and Characterization of Hetero-bifunctional Cross-linking Reagents for Protein Modifications, Chem. Pharm. Bull., 1981, 29, 1130.3. S. Yoshitake, M. Imagawa, E. Ishikawa, Y. Niitsu, I. Urushizaki, M. Nishimura, R. Kanazawa, H. Kurosaki, S. Tachibana, N. Nakazawa and H. Ogawa, Mild and Efficient Conjugation of Rabbit Fab Eand Horseradish Peroxidase Using a Maleimide Compound and Its Use for Enzyme Immunoassay, J. Biochem., 1982, 92, 1413.4. E. Ishikawa, M. Imagawa, S. Hashida, S. Yoshitake, Y. Hamaguchi and T. Ueno, Enzyme-Labelling of Antibodies and Their Fragments for Enzyme Immunoassay and Immunohistochemical Staining, J. Immunol., 1983, 4, 209.5. I. Weeks, A. K. Campbell and J. S. Woodhead, Two-site Immunochemiluminometric Assay for Human α1-Fetoprotein, Clin. Chem., 1983, 29, 1480.6. S. Hashida, M. Imagawa, S. Inoue, K. Ruan and E. Ishikawa, More Useful Maleimide Compounds for the Conjugation of Fab Eto Horseradish Peroxidase through Thiol Groups in the Hinge, J. Appl. Biochem., 1984, 6, 56.7. S. Inoue, M. Imagawa, S. Hashida, K. Ruan and E. Ishikawa, A Small Scale Preparation of Affinity-Purified Rabbit Fab EHorseradish Peroxidase Conjugate for Enzyme Immunoassay, Anal. lett., 1984, 17, 229.8. M. Koizumi, K. Endo, M. Kunimatsu, H. Sakahara, T. Nakashima, Y. Kawamura, Y. Watanabe, T. Saga, J. Konishi, T. Yamamura, S. Hosoi, S. Toyama, Y. Arano and A. Yokoyama, Ga-Labeled Antibodies for Immunoscintigraphy and Evalution of Tumor Targeting of Drug-Antibody Conjugatesin Mice, Cancer Res., 1988, 48, 1189.9. T. Kohno and E. Ishikawa, Novel Enzyme Immunoassay(Immune Complex Transfer Enzyme Immunoassay) for Anti-insulin IgG in Guinea Pig Serum, Anal. lett., 1988, 21, 1019.

Dojindo,Biotin-AC5 Sulfo-OSu/10/B320,抗生物素蛋白-生物素系统

Product Description of Amine-Reactive Biotins
抗生物素蛋白-生物素系统在免疫学和组织化学中有许多应用。抗生物素蛋白和生物素之间的相互作用非常强，解离常数约为 10-15 M。生物素通常添加到一抗或二抗中，例如抗 IgG 和抗 IgM。在用生物素标记的抗体制备抗原-抗体复合物后，使用酶或荧光素标记的抗生物素蛋白或链霉抗生物素蛋白对抗原进行比色或荧光检测。琥珀酰亚胺酯生物素在 pH 7-9 下与伯胺和仲胺（例如氨基酸和蛋白质）反应。琥珀酰亚胺酯与游离胺基反应生成稳定的酰胺键。琥珀酰亚胺基生物素试剂必须溶解在 DMSO、DMF 或酒精中。用 DMSO 制备的储备溶液在 -20ºC 下可稳定数月。磺基琥珀酰亚胺生物素试剂可溶于水，因此无需使用 DMF 或 DMSO 等有机溶剂。使用具有较长间隔物的生物素（如 Biotin-(AC5)2-OSu 或 Biotin-(AC5)2-Sulfo-OSu）制备的 IgG 具有更好的信噪比。较长的间隔使链霉亲和素或抗生物素 IgG 能够在没有结构抑制的情况下识别生物素。因此，Biotin-(AC5)2-OSu 被用作 Biotin Labeling Kit-NH2 中的生物素标记剂。

Labeling Procedure for IgG1. Prepare 10 mM of the biotin labeling reagent using DMSO.2. Prepare 100 μl of 1 mg per ml IgG buffer solution (pH 7.5-8.5) that does not contain any large molecules with amine compounds.3. Add 1-5ml biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37ºC for 1 hour.4. Remove excess biotin labeling reagent using gel filtration or dialysis.5. Prepare solutions for further experiment using an appropriate buffer such as PBST (0.05% Tween 20/PBS).

1. J. Wormmeester, F. Stiekema and C. Groot, Immunoselective Cell Separation, Methods Enzymol., 1990, 184, 314.2. J. J. Leary, D. J. Brigati and D. C. Ward, Rapid and Sensitive Colorimetric Method for Visualizing Biotin-labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-blots, Proc. Batl. Acad. Sci. USA, 1983, 80, 4045.3. W. T. Lee and D. H. Conrad, The Murine Lymphocyte Receptor for IgE. II. Characterization of the Multivalent Nature of the B Lymphocyte Receptor for IgE, J. Exp. Med., 1984, 159, 1790.4. D. R. Gretch, M. Suter and M. F. Stinski, The use of Biotinylated Monoclonal Antibodies and Streptavidin Affinity Chromatography to Isolate Herpesvirus Hydrophobic Proteins or Glycoproteins, Anal. Biochem., 1987, 163, 270 .5. M. Shimkus, J. Levy and T.Herman, A Chemically Cleavable Biotinylated Nucleotide: Usefulness in the Recovery of Protein-DNA Complexes from Avidin Affinity Columns, Proc. Natl. Acad. Sci. USA, 1985, 82, 2593.6. W. J. LaRochelle and S. C. Froehner, Immunochemical Detection of Proteins Biotinylated on Nitrocellulose Replicas, J. Immunol. Methods, 1986, 92, 65.7. P. S. Anjaneyulu and J. V. Staros, Reactions of N-hydroxysulfosuccinimide Active Esters, Int. J. Pep. Protein Res., 1987, 30, 117.8. H. M. Ingalls, C. M. Goodloe-Holland and E. J. Luna, Junctional Plasma Membrane Domains Isolated from Aggregating Dictyostelium Discoideum Amebae, Proc. Natl. Acad. Sci. USA, 1986, 83, 4779.9. J. L. Guesdon, T. Ternynck and S. Avrameas, The Use of Avidin-biotin Interaction in Immunoenzymatic Techniques, J. Histochem, Cytochem, 1979, 27, 1131.

Dojindo,Biotin-HPDP/50/B573,生物素-HPDP含有吡啶基二硫基的生物素分子

生物素-HPDP 是一种含有吡啶基二硫基的生物素分子。 Biotin-HPDP可以将生物素引入到含有SH基团的分子中。 吡啶基二硫基也可以很容易地被还原剂还原，与这类蛋白质的SH基团发生交换反应是可能的。 与 Biotin-SS-Sulfo-OSu 一样，通过还原操作的接头位点具有可切割的特性。但是，由于它与目标蛋白质的 SH 基团反应，因此反应位点可以在还原后返回到源自原始蛋白质的 SH 基团。 二硫基。 在涂有抗生物素蛋白或链霉抗生物素蛋白的柱上使用生物素-HPDP标记分子后，可以通过还原操作裂解连接位点来回收分子。

1. A.Janshoff, K-P. S. Dancil, C. Steinem, D. P.Greiner, V. S.-Y.Lin, C. Gurtner, K.Motesharei, M. J. Sailor and G. M. R. Ghadiri, “Macroporous p-Type SiliconFabry-Perot Layers. Fabrication, Characterization, and Applications inBiosensing”, J. Am. Chem.Soc., 1998, 120(46), 12108.2. K. K.Caswell, J. N. Wilson, U. H. F. Bunz and C. J. Murphy, “PreferentialEnd-to-end Assembly of Gold Nanorods by Biotin-streptavidin Connectors”, J.Am. Chem.Soc., 2003, 125(46), 13914.3. Z. Siwy, L. Trofin, P. Kohli, L. A.Baker, C. Trautmann and C. R. Martin,”Protein Biosensors Based on Biofunctionalized Conical GoldNanotubes”, J. Am. Chem.Soc., 2005, 127(14), 5000.4. G. Hao, B. Derakhshan, L. Shi, F. Campagne and S. S. Gross, “SNOSID, AProteomic Method for Identification of Cysteine S-nitrosylation Sites inComplex Protein Mixtures”, Proc. Natl. Acad. Sci. U. S. A., 2006,103(4), 1012.5. N. L. Moan, G. Clement, S. L. Maout, F. Tacnet and M. B. Toledano, “TheSaccharomyces Cerevisiae Proteome of Oxidized Protein Thiols: ContrastedFunctions for the Thioredoxin and Glutathione Pathways”, J. Biol. Chem.,2006, 281(15), 10420.6. W. Shu, E. D. Laue and A. A. Seshia, “Investigation ofBiotin-streptavidin Binding Interactions Using Microcantilever Sensors”, Biosens.Bioelectron., 2007, 22, 2003.7. W. Yang, D. D. Vizio, M. Kirchner, H. Steen and M. R. Freeman,”Proteome Scale Characterization of Human S-acylated Proteins inLipid Raft-enriched and Non-raft Membranes”, Mol. Cell. Proteomics,2010, 9, 54.

Dojindo,Sulfo-SMCC/50/S330,Sulfo-SMCC 是一种双功能试剂

Sulfo-SMCC 是一种双功能试剂，在分子的不同末端含有一个马来酰亚胺基团和一个 N-羟基琥珀酰亚胺活性酯。 马来酰亚胺基团与SH基团反应，但N-羟基琥珀酰亚胺活性酯与氨基特异性反应。 在pH高于中性时，活性酯基与氨基形成稳定的酰胺键。 另一方面，马来酰亚胺基可以在pH6-7的条件下选择性地与硫醇基反应。 此外，具有环己烷结构的接头部位的磺基-SMCC的特征在于，与在接头部位具有芳香环的同类交联剂相比，马来酰亚胺基团的稳定性提高。 由于导入了具有磺酸基的活性酯基，因此可以在不使用DMSO、DMF等有机溶剂的情况下进行标记反应。

Hetero-bifunctional Reagents

Product Name Code Length (Å)
Sulfo-EMCS S024 9.4
Sulfo-GMBS S025 6.9
Sulfo-HMCS S026 13.0
Sulfo-KMUS S250 16.7
Sulfo-SMCC S330 8.0
1. S. Hashida, M. Imagawa, S. Inoue, K.-H. Ruan and E. Ishikawa, “MoreUseful Maleimide Compounds for the Conjugation of Fab’ to HorseradishPeroxidase Through Thiol Groups in the Hinge”, J. Appl. Biochem., 1984,6, 56.2. O. Prat, E. Lopez and G. Mathis, “Europium(III) Cryptate: A FluorescentLabel for the Detection of DNA Hybrids on Solid Support”, Anal. Biochem.,1991, 195(2), 283.3. O. Siiman, A. Burshteyn, J. A. Maples and J. K. Whitesell,”Tris(3-mercaptopropyl)-N-glycylaminomethane as a New Linker to BridgeAntibody with Metal Particles for Biological Cell Separations”, BioconjugateChem., 2000, 11(4), 549.4. T. P. Thomas, A. K. Patri, A. Myc, M. T. Myaing, J. Y. Ye, T. B. Norris andJ. R. Baker Jr., “In Vitro Targeting of Synthesized Antibody-conjugatedDendrimer Nanoparticles”, Biomacromolecules, 2004, 5(6),2269.5) V. Gauvreau, G. Laroche, “Micropattern Printing of Adhesion, Spreading,and Migration Peptides on Poly(tetrafluoroethylene) Films to PromoteEndothelialization”, Bioconjugate Chem., 2005, 16(5),1088.

Dojindo,MitoPeDPP/1/M466,MitoPeDPP 是一种细胞膜可渗透探针

MitoPeDPP 是一种细胞膜可渗透探针，基于苝的染料。 由于引入了三苯基鏻部分，它特别定位于线粒体。 MitoPeDPP 用于监测铁死亡研究中的脂质过氧化。由于 MitoPeDPP 的激发和发射波长分别为 452 nm 和 470 nm，该探针可用于活细胞中的亲脂性过氧化物成像。 Shioji 等人。 福冈大学化学系

Lipophilic Peroxide Detection in Mitochondria (cell line used: HepG2 cell)

A: MitoPeDPP stained Mitochondria with t-BHP treatmentB: MitoRed stained MitochondriaC: Merged Image (A/B)

Selectivity of MitoPeDPP as a Peroxides Probe Even though MitoPeDPP reacts with various peroxides (H2O2, t-BHP, ONOO-) in homogeneous systems (data is not shown), the MitoPeDPP is specifically-oxidized by t-BHP in mitochondria (A) but not with ROS and RNS (B).

A: MitoPeDPP stained cells with t-BHP treatment (t-BHP) and without (control).B: MitoPeDPP stained cells with ROS or RNS exposure.*As ROS generators, PMA (O2–・), NOC7 (NO), and SIN-1 (ONOO–) were used in the experiments.*Abbreviationst-BHP: tert-butylhydroperoxidePMA: Phorbol Myristate AcetateNOC7: 1-Hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazineSIN-1: 3-(Morpholinyl)sydnonimine, hydrochloride

Dojindo,SPDP/100/S291,SPDP 是一种异双功能试剂

SPDP 是一种异双功能试剂，可用于桥接两种不同的蛋白质，例如酶和抗体。 SPDP 首先通过其氨基与蛋白质分子反应。 SPDP 将吡啶基二硫化物部分引入蛋白质，然后被 DTT 还原以形成硫醇基团。 然后这些硫醇基团与另一个蛋白质分子形成二硫键，从而产生异二聚体蛋白质。
Structural Formula

Homo-bifunctional Reagents

Product Name Code Length (Å) Solvent
DSP D629 8.5 DMSO, DMF
SPDP S291 4.1 Acetonitrile

1. P. Walden, Z. A. Nagy and J. Klein, Major Histocompatibility Complex-Restricted and Unrestricted Activation of Helper T Cell Lines by Liposome-bound Antigens, J. Mol. Cell. Immunol, 1986, 2, 191.2. R. D. Gordon, W. E. Fieles, D. L. Schotland, R. Hogue-Angeletti and R. L. Barchi, Topographical Localization of the C-terminal Region of the Voltagedependent Sodium Channel from Electrophorus Electricus Using Antibodies Raised Against a Synthetic Peptide, Proc. Natl. Acad. Sci. USA, 1987,84, 308.

Dojindo,GMBS/100/G005,杂双功能交联剂具有活化的酯和马来酰亚胺反应基团

杂双功能交联剂具有活化的酯和马来酰亚胺反应基团。 这些官能团分别与蛋白质的胺和巯基反应。 酶标记的半抗原是使用异双功能交联剂（例如 EMCS 或 GMBS）制备的。 交联反应需要中性 pH 值和温和的温度，因为在交联反应中需要保持酶活性和抗体滴度。 可使用具有 3、5、7 或 10 个线性碳链的异双功能交联剂。 这些线性脂肪链充当两个反应位点与其水溶性试剂之间的间隔物。 在更宽的 pH 范围内，它们比芳香族交联剂（例如 succinimidyl-4-N-maleimidobenzoate）更稳定。

Conjugation of Macromolecules with Hetero-Bifunctional Cross-Linking Reagent

Hetero-bifunctional Reagents

Product Name Code Length (Å) Solvent
EMCS E018 9.4 DMSO
GMBS G005 6.9 Chloroform, DMF
HMCS H257 13.0 Acetonitrile
KMUS K214 16.7 Acetonitrile, Methyl alcohol

 

1. S. Yoshitake, Y. Yamada, E. Ishikawa and Rene Masseyeff, Conjugation of Glucose Oxidase from Aspergillus Niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide, Eur. J. Biochem., 1979, 101, 395.2. H. Tanimori, T. Kitagawa, T. Tsunoda and R. Tsuchiya, Enzyme Immunoassay of Neocarzinostain Using β-Galactosidase as Label, J. Pharm. Dyn., 1981, 4, 812.3. K. Fujiwara, M. Yasuno and T. Kitagawa, Novel Preparation Method of Immunogen for Hydrophobic Hapten, Enzyme Immunoassay for Daunomycin and Adriamycin, J. Immunol. Methods, 1981, 45, 195.4. T. Miura, H. Kouno and T. Kitagawa, Detection of Residual Penicillin in Milk by Sensitive Enzyme Immunoassay, J. Pharm. Dyn., 1981, 4, 706.5. S. Yoshitake, M. Imagawa, E. Ishikawa, Y. Niitsu, I. Urushizaki, M. Nishiura, R. Kanazawa and H. Ogawa, Mild and Efficient Conjugation of Rabbit Fab Eand Horseradish Peroxidase Using a Maleimide Compound and Its Use for Enzyme Immunoassay, J. Biochem. ,1982, 92, 1413.6. T. Kitagawa, H. Tanimori, K. Yoshida, H. Asada, T. Miura and K. Fujiwara, Studies on Viomycin. XV. Comparative Study on the Specificities of Two Anti-viomycin Antisera by Enzyme Immunoassay, Chem. Pharm. Bull., 1982, 30, 2487.7. T. Kitagawa, T. Kawasaki and H. Munechika, Enzyme Immunoassay of Blasticidin S with High Sensitivity:A New and Convenient Method for Preparation of Immunogenic(Hapten-Protein) Conjugates, J. Biochem.,1982, 92, 585.

Dojindo,Alkaline Phosphatase Labeling Kit-SH/3/LK13,碱性磷酸酶标记试剂盒-SH

碱性磷酸酶标记试剂盒-SH 主要用于制备碱性磷酸酶标记的 IgG 用于酶免疫测定 (EIA) 和制备碱性磷酸酶标记的抗原用于竞争性 EIA。 SH-reactive ALP 是该试剂盒的一个组成部分，可以与蛋白质或其他分子的硫醇基团发生反应（图 1）。 该试剂盒包含标记过程所需的所有试剂，包括还原剂和储存缓冲液。 SH 反应性 ALP 与靶分子形成共价键。 还原剂可以在 IgG 分子中产生游离硫醇基团。 当碱性磷酸酶标记的 IgG 用于 EIA 时，SH 反应性 ALP 的标记效率足以消除标记后的任何纯化过程。 如果标记后需要高纯度偶联物，只需使用亲和柱或凝胶渗透柱即可。 标记小分子时，可以使用该套件中包含的过滤管去除多余的分子。

Precaution♦ The molecular weight of the reduced protein to be labeled with this kit should be greater than 50,000.♦ The molecular weight of the small amine compound to be labeled with this kit should be smaller than 5,000.♦ IgG or alkaline phosphatase-conjugated IgG is always on the membrane of the filtration tube during the labeling process.♦ If the IgG solution contains other proteins with molecular weight larger than 10,000, such as BSA or gelatin, purify the IgG solution prior to labeling alkaline phosphatase with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

Peroxidase Labeling Kit-NH2-used for Viral Research (COVID-19 Testing)

Dr. Kyosei et al. developed the ultrasensitive detection of spike proteins of SARS-CoV-2. The SARS-CoV-2 spike proteins were measured by a sandwich ELISA coupled with thio-NAD cycling using alkaline phosphatase, androsterone derivatives, and 3α-hydroxysteroid dehydrogenase (3α-HSD) and its coenzymes (NADH and thio-NAD). The secondary antibody was conjugated to alkaline phosphatase by using Dojindo’s Alkaline Phosphate Labeling Kit-SH.

Y. Kyosei et al., Diagnostics, 2020, 10(8), 594.“Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2”

Sandwich ELISA

Fig. 2 Sandwich ELISA of human TNF-α detection

Plate: 2μg/ml anti-human TNF-antibody (rabbit, polyclonal)-coatedhigh binding platerecombinant human TNF-a: 0-1000 pg/ml PBSTALP-conjugated anti-human TNF-antibody: Prepared by Alkaline phosphatase Labeling Kit-SH .1μg/ml PBST+blocking reagentSubstrate: p-Nitrophenylphosphate, ALP substrate

Can I use this kit for F(ab E2?
Yes, please follow the labeling protocol for IgG. The recovery of the conjugate should be over 80%.
Can I use this kit for other proteins or peptides?
Yes, if the molecular weight of the reduced form is greater than 50,000 or less than 5,000, and it has a reactive SH group or a disulfide group that can be reduced without losing activity. If the molecular weight is greater than 50,000, follow the labeling protocol for IgG and use 0.5-1 nmol of sample protein for LK13-10. If the molecular weight is less than 5,000, follow the labeling protocol for small molecules. If the molecular weight is between 5,000 and 50,000, contact our customer service at info@dojindo.com or 1-877-987-2667 for more information.
Can I use this kit to label oligopeptides or oligonucleotides?
Yes, if the molecular weights of the oligonucleotide or the oligopeptide are less than 5,000 and they have at least one SH group. Follow the labeling protocol for small molecule.
What is the minimum amount of IgG that can be labeled with LK13-10?
The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG. However, even 10 μg IgG can be labeled, using 1/5 volume of SH-reactive ALP solution at step 8.
How many alkaline phosphatase molecules per reduced IgG are introduced?
The average number of alkaline phosphatase molecule per reduced IgG is 1 to 2.
Do I have to use a Filtration tube prior to labeling the protein?
If the protein solution does not contain small molecules with reactive SH groups and the concentration of the protein is 10 mg per ml, or about 70 μM, there is no need to use the Filtration tube. Just mix 10 μl of the sample solution with Solution B and add the mixture to a vial of the SH-reactive peroxidase.
Do I have to use Storage buffer included with the kit?
No, you don’t have to use Storage buffer from the kit. You can choose any kind of buffer appropriate for your experiment.
My sample contains small insoluble material. What should I do?
Spin the sample and use the supernatant for labeling.
Does unconjugated SH-reactive ALP still have a reactive maleimide after the labeling reaction to IgG?
No. Nearly 100% of SH-reactive ALP is used for the IgG labeling or the small molecule labeling.
Does Storage buffer contain animal products or polymers?
No, Storage buffer does not contain any animal products , polymers, or heavy metal ions.

Dojindo,KMUS/50/K214,杂双功能交联剂具有活化的酯和马来酰亚胺反应基团

杂双功能交联剂具有活化的酯和马来酰亚胺反应基团。 这些官能团分别与蛋白质的胺和巯基反应。 酶标记的半抗原是使用异双功能交联剂（例如 EMCS 或 GMBS）制备的。 交联反应需要中性 pH 值和温和的温度，因为在交联反应中需要保持酶活性和抗体滴度。 可使用具有 3、5、7 或 10 个线性碳链的异双功能交联剂。 这些线性脂肪链充当两个反应位点与其水溶性试剂之间的间隔物。 在更宽的 pH 范围内，它们比芳香族交联剂（例如 succinimidyl-4-N-maleimidobenzoate）更稳定。

Conjugation of Macromolecules with Hetero-Bifunctional Cross-Linking Reagent

Hetero-bifunctional Reagents

Product Name Code Length (Å) Solvent
EMCS E018 9.4 DMSO
GMBS G005 6.9 Chloroform, DMF
HMCS H257 13.0 Acetonitrile
KMUS K214 16.7 Acetonitrile, Methyl alcohol

 

Dojindo,ExoSparkler 外泌体膜标记试剂盒/Deep/EX01,ExoSparkler Exosome Membrane Labeling Kit

外泌体是细胞外囊泡 (EV) 的一种形式，可能导致癌症的恶性转化和转移。 因此，通过外泌体进行的细胞间通讯引起了科学界的极大兴趣。 为了阐明这种交流，已经使用了基于荧光染料的标记技术。 标记细胞膜的荧光染料通常用于外泌体标记，因为外泌体中的脂质双层是合适的标记目标。 ExoSparkler 系列可用于对纯化的外泌体膜或蛋白质进行染色，从而可以对细胞摄取的标记外泌体进行成像。

ExoSparkler series enables you to observe exosome dynamics more accurately.Commonly used exosomal membrane dye can cause dye aggregation, exhibiting fluorescent spots that are not derived from exosomes. These dyes can also change the functional properties of exosomes while increasing the background imaging.1,2The dyes used in ExoSparkler series (Mem Dye-Green, Red, and Deep Red) do not cause aggregation and have little influence on properties of exosomes, allowing a more accurate observation of exosome dynamics.1) Mehdi Dehghani et al., “Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size”.bioRxiv., 2019, doi:10.1101/532028.2) Pužar Dominkuš P et al., “PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles.” Biochim Biophys Acta Biomembr., 2018, doi: 10.1016/j.bbamem.2018.03.013.

ExoSparkler series does not allow extracellular aggregation

Exosomes stained with ExoSparkler’s Mem Dye-Deep Red or an alternative product (green or red) were added to each well containing HeLa cells. The labeled exosomes taken into HeLa cells were observed by fluorescent microscopy. As a result, extracellular fluorescent spots suspected of dye aggregations were seen in each well containing the exosomes stained with the alternative product (green or red).

Experimental conditionsExosomes were purified by ultracentrifugation (10μg exosome protein) and stained with each dye. Labeled exosomes were added to HeLa cells (1.25×104 cells), and the cells were incubated for 24 hours. Cells were washed, and immunofluorescence images showing labeled exosomes were observed.Detection conditionsMem Dye-Deep Red(Purple): Ex 640nm/Em 640-760nmAlternative Product “P” (Green): Ex 561nm/Em 560-620nmAlternative Product “P” (Red): Ex 640nm/Em 650-700nm

Mem Dye-Deep Red and Product “P” (Green and Red) in aqueous solution were analyzed by NTA (nanoparticle tracking analysis) to investigate the generation of aggregates. No aggregation was observed in the experiments with Mem Dyes, although Product “P” (Green and Red) produced dye-to-dye aggregates (100–500 nm size).Instrument: LM10-HSBFT 14 (Nanosight)

Change the particle size of Mem Dye-solution and Product “P” solution

In Mem Dye-Green, Red, the aggregation of the dye was not confirmed as in Mem Dye-Deep Red.

Mem Dyes have little effect on exosome propertiesNTA (nanoparticle tracking analysis) and zeta potential were measured to determine the changes in exosomes of dye-stained with Mem Dye-Deep Red or Product “P” (green or red) or unstained exosomes.As a result, the Mem-Dye series (green, red, deep red) had little effect on exosome properties.

Effect of the dyes on the particle size of the exosomesExosomes were stained with Mem Dye-series (green, red, deep red) and Product “P” (green and red) at a dye concentration of 10 μmol/L in DMSO, the NTA (nanoparticle tracking analysis) of the stained exosomes (as 10 µg protein) was measured.As a result, Mem Dyes-series did not change number and particle size of the exosomes (bottom left). Conversely, the Product “P” stained exosomes showed the significant changes of particle size and population of the exosomes (bottom right).Instrument: LM10-HSBFT 14 (Nanosight)

Effect of the dyes on the zeta potentials of the exosomesExosomes were stained with Mem Dye-series (green, red, crimson) and Product “P” (green and red) at a dye concentration of 10 μmol/L in DMSO, the zeta potentials of the stained exosomes (as 10 µg protein) was measured.As a result, product “P”-stained exosomes have lower zeta potential than Mem Dye-stained.Instrument: Zetasizer Nano ZSP (Malvern Panalytical)

Zeta potentials comparison of dye-stained (Mem-Dye/Product “P”) or unstained exosomes

References) Takashi Shimomura et al., “New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes”.bioRxiv., 2020, doi:10.1101/2020.02.02.931295.

Our ExoSparkler Exosome Membrane Labelling Kits provide everything from fluorescence labeling to purificationExoSparkler series contains filtration tubes available for the removal of dyes unreacted after fluorescence labeling, as well as an optimized protocol for labeling exosomes. Our ExoSparkler series makes it possible to prepare fluorescence labeling of exosomes using the simple procedure.

Comparison of purification methods (removal of unlabeled dyes)The filtration tubes used to remove unlabeled dyes in this kit can purify exosomes at a higher recovery rate than gel filtration methods.

For the effectiveness of purification using filtration tubes, please see Q&A.(The filter is colored in the purification after the labeling, Have unlabeled dyes been removed?)

Observe the time-dependent changes in exosomes localization

<Experimental conditions>Exosomes purified by ultracentrifugation (10 µg as protein amount) were stained with Mem Dye-Deep Red (Exosome Membrane Fluorescence Labeling Kit) and added to HeLa cells (1.25×104 cells) stained with lysosome staining dye. The fluorescence images were observed after 1 h and 4 h incubation.As a result, it was confirmed that the fluorescence puncta (purple) of Mem Dye-Deep Red overlapped with the localization of lysosomes (green) over time (white), and that the localization of exosomes changed in a time-dependent manner.

 

<Detection Conditions>Mem Dye-Deep Red (Purple): Ex 640 nm/Em 640-760 nmLysosome staining dye: Ex 488 nm/Em 490-540 nm

 

Visualization of EVs uptake via endocytic pathway

 

Mem Dye-labeled EVs are internalized via endocytosis:HeLa cells were incubated with 10 μmol/L ECGreen (Code: E296) for 30 min. Then, Mem Dye-Deep Red labeled EVs (quantified as 10 µg of protein) were added to HeLa cells. After 30 or 120 min incubation, the cells were washed and observed under a fluorescence microscope (Scale Bar: 10 µm).

 

ExoSparkler series product comparison

Experimental conditionsExosomes were purified by ultracentrifugation (10 μg exosome protein) and stained with each dye. Labeled exosomes were added to HeLa cells (1.25×104 cells), and the cells were incubated for 24 hours. Cells were washed, and immunofluorescence images showing labeled exosomes were observed.Detection conditionsGreen: Ex 488nm/Em 490-540nmRed: Ex 561nm/Em 570-640nmDeep Red: Ex 640nm/Em 640-760nm

Are there recommended purification methods for exosome?
We recommended the exosomes prepared by ultracentrifuge, moreover we had evaluated the exosomes prepared by immunoprecipitation.Also, exosome prepared by polymer-based precipitation cannot be applied with these kits.
Some dye remains on the filter after purification. Have unlabeled dyes been removed?

Unlabeled dye is likely to be retained inside the filter, but we have confirmed that the unlabeled dye is not remaining on top of the filter. Please refer to the following experiment:<Experimental condition>① We stained the 10 µg exosomes (as protein amount) purified by ultracentrifugation.② We also prepared only using buffer as a control3. The recovered solution is added to HeLa cells (1.25 x 104 cells), and the fluorescence image was observed 4 hours later.As a result, it was confirmed that no bright fluorescent particles (unlabled dyes) were observed in the cells using the recovered solution with the only buffer.① Exosome + Buffer

② Only Buffer

Fluorescent images at 4 h incubation

Detection conditionsGreen：Ex 488 nm / Em 490 – 540 nmRed ：Ex 561 nm / Em 570 – 640 nmDeep Red：Ex 640 nm / Em 640 – 760 nm

Can I store the exosomes after it has been stained?

We can not recommend to store the exosomes after it has been stained.Please use the stained exosomes as soon as possible.

Which media have been used in exosome uptake experiments?
We had evaluated the exosome uptake experiments using MEM (Minimum Essential Medium) and DMEM (Dulbecco’s Modified Eagle’s Medium). We can not recommend to use the serum-free medium because this kit has been optimized to observe in serum-containing medium.

Dojindo,生物素-（AC5）2磺基OSu/10/B321

Product Description of Amine-Reactive Biotins
抗生物素蛋白-生物素系统在免疫学和组织化学中有许多应用。抗生物素蛋白和生物素之间的相互作用非常强，解离常数约为 10-15 M。生物素通常添加到一抗或二抗中，例如抗 IgG 和抗 IgM。在用生物素标记的抗体制备抗原-抗体复合物后，使用酶或荧光素标记的抗生物素蛋白或链霉抗生物素蛋白对抗原进行比色或荧光检测。琥珀酰亚胺酯生物素在 pH 7-9 下与伯胺和仲胺（例如氨基酸和蛋白质）反应。琥珀酰亚胺酯与游离胺基反应生成稳定的酰胺键。琥珀酰亚胺基生物素试剂必须溶解在 DMSO、DMF 或酒精中。用 DMSO 制备的储备溶液在 -20ºC 下可稳定数月。磺基琥珀酰亚胺生物素试剂可溶于水，因此无需使用 DMF 或 DMSO 等有机溶剂。使用具有较长间隔物的生物素（如 Biotin-(AC5)2-OSu 或 Biotin-(AC5)2-Sulfo-OSu）制备的 IgG 具有更好的信噪比。较长的间隔使链霉亲和素或抗生物素 IgG 能够在没有结构抑制的情况下识别生物素。因此，Biotin-(AC5)2-OSu 被用作 Biotin Labeling Kit-NH2 中的生物素标记剂。

Labeling Procedure for IgG1. Prepare 10 mM of the biotin labeling reagent using DMSO.2. Prepare 100 μl of 1 mg per ml IgG buffer solution (pH 7.5-8.5) that does not contain any large molecules with amine compounds.3. Add 1-5ml biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37ºC for 1 hour.4. Remove excess biotin labeling reagent using gel filtration or dialysis.5. Prepare solutions for further experiment using an appropriate buffer such as PBST (0.05% Tween 20/PBS).

1. J. Wormmeester, F. Stiekema and C. Groot, Immunoselective Cell Separation, Methods Enzymol., 1990, 184, 314.2. J. J. Leary, D. J. Brigati and D. C. Ward, Rapid and Sensitive Colorimetric Method for Visualizing Biotin-labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-blots, Proc. Batl. Acad. Sci. USA, 1983, 80, 4045.3. W. T. Lee and D. H. Conrad, The Murine Lymphocyte Receptor for IgE. II. Characterization of the Multivalent Nature of the B Lymphocyte Receptor for IgE, J. Exp. Med., 1984, 159, 1790.4. D. R. Gretch, M. Suter and M. F. Stinski, The use of Biotinylated Monoclonal Antibodies and Streptavidin Affinity Chromatography to Isolate Herpesvirus Hydrophobic Proteins or Glycoproteins, Anal. Biochem., 1987, 163, 270 .5. M. Shimkus, J. Levy and T.Herman, A Chemically Cleavable Biotinylated Nucleotide: Usefulness in the Recovery of Protein-DNA Complexes from Avidin Affinity Columns, Proc. Natl. Acad. Sci. USA, 1985, 82, 2593.6. W. J. LaRochelle and S. C. Froehner, Immunochemical Detection of Proteins Biotinylated on Nitrocellulose Replicas, J. Immunol. Methods, 1986, 92, 65.7. P. S. Anjaneyulu and J. V. Staros, Reactions of N-hydroxysulfosuccinimide Active Esters, Int. J. Pep. Protein Res., 1987, 30, 117. 8. H. M. Ingalls, C. M. Goodloe-Holland and E. J. Luna, Junctional Plasma Membrane Domains Isolated from Aggregating Dictyostelium Discoideum Amebae, Proc. Natl. Acad. Sci. USA, 1986, 83, 4779.9. J. L. Guesdon, T. Ternynck and S. Avrameas, The Use of Avidin-biotin Interaction in Immunoenzymatic Techniques, J. Histochem, Cytochem, 1979, 27, 1131.

Dojindo,异硫氰酸苄基EDTA/10/M030

Product Description of Amine-Reactive Biotins
Isothiocyanobenzyl-EDTA 通过其胺基将 EDTA 部分添加到蛋白质中。 然后可以用重金属离子和放射性金属（如 111In 和 99Tc）标记 EDTA 标记的蛋白质，并用于免疫成像。

Chemical Structure

1. C. F. Meares and T. G. Wensel, Metal Chelates as Probes of Biological Systems, Acc. Chem. Res.,1984, 17, 202.2. S. V. Deshpande, R. Subramanian, M. J. McCall, S. J. Denardo, G. L. Denardo and C. F. Meares, Metabolism of Indium Chelates Attached to Monoclonal Antibody: Minimal Transchelation of Indium form Benzyl-EDTA Chelate in vivo, J. Nucl. Med., 1990, 31, 218.3. S. Mirzadeh, M. W. Brechbiel and R. W. Atcher, Otto A. Gansow Radiometal Labeling of Immunoproteins: Covalent Linkage of 2-(4-Isothiocyanatobenzyl) diethylenetriaminepentaacetic acid Ligands to Immunoglobulin, Bioconjugate Chem., 1990, 1, 59. 

Dojindo,Biotin-SS-Sulfo-OSu/100/B572,Biotin-SS-Sulfo-Osu有N-羟基琥珀酰亚胺活性酯基团生物素分子

Biotin-SS-Sulfo-Osu是一种含有N-羟基琥珀酰亚胺活性酯基团的生物素分子，可用于将生物素引入抗体、酶等蛋白质的氨基基团。 与仅具有烷基链接头的生物素化试剂、Biotin-AC5Sulfo-OSu 或 Biotin Sulfo-OSu 不同，接头位点的切割是可能的，但是引入接头位点的二硫基团很容易被还原剂还原。 在涂有抗生物素蛋白或链霉抗生物素蛋白的柱上使用 Biotin-SS-Sulfo-OSu 标记分子后，可以通过还原操作切割接头位点来回收分子。

1. M. Shimkus, J. Levy and T.Herman, “A Chemically Cleavable Biotinylated Nucleotide: Usefulness in theRecovery of Protein-DNA complexes From Avidin Affinity Columns”, Proc.Natl. Acad. Sci. USA, 1985, 82, 2593.2. G. Nikov, V. Bhat, J. S. Wishnok and S. R. Tannenbaum, “Analysis ofNitrated Proteins by Nitrotyrosine-specific Affinity Probes and MassSpectrometry”, Anal. Biochem., 2003, 320(2), 214.3. S. B. Scheurer, C. Roesli, D. Neri and G. Elia, “A Comparison ofDifferent Biotinylation Reagents, Tryptic Digestion Procedures, and MassSpectrometric Techniques for 2-D peptide Mapping of Membrane Proteins”, Proteomics,2005, 5, 3035.4. J. C. Timmer, M. Enoksson, E. Wildfang, W. ZHU, Y. Igarashi, J-B. Denault,Y. Ma, B. Dummitt, Y-H. Chang, A. E. Mast, A. Eroshkin, J. W. Smith, W. A. Taoand G. S. Salvesen, “Profiling Constitutive Proteolytic events in vivo”,Biochem. J., 2007, 407, 41.5. S. J. Lee, K-H. Kim, J. S. Park, J. W. Jung, Y. H. Kim, S. K. Kim, W-S. Kim,H-G. Goh, S-H Kim, J-S. Yoo, D-W Kim and K. P. Kim, “Comparative Analysisof Cell Surface Proteins in Chronic and Acute Leukemia Cell Lines”, Biochem.Biophys. Res. Commun., 2007, 357, 620.

Dojindo,Ab-10 Rapid Peroxidase Labeling Kit/3/LK33,Ab-10 快速过氧化物酶标记试剂盒

Ab-10 快速过氧化物酶标记试剂盒用于在 30 分钟内将过氧化物酶标记为 10 µg 抗体。 试剂盒中的标记剂具有琥珀酰亚胺酯基团，无需任何活化过程即可轻松与靶抗体的氨基形成共价键。 该试剂盒包含抗体标记所需的所有试剂。

IgG Labeling Reaction of NH2-reactive Peroxidase

Labeing Procedure

Precaution♦ Use 0.5-1 mg/ml of antibody solution for labeling. If the antibody concentration is higher than 1mg/ml, dilute the antibody solution with PBS.♦ If the sample solution contains small insoluble materials, centrifuge the solution, and use the supernatant for the labeling.♦ The microtube in this kit contain solutions. Since there is a possibility that the droplets might be attached to the inside walls or caps, please centrifuge to drop the droplets prior to opening.♦ Some additives in antibody solution may interfere with the labeling if the concentration is too high. The maximum compatible concentrations of such additives are indicated on Table 1.♦ After a Reactive Fluorescent is removed from the seal bag, keep the unused Reactive Fluorescein in the bag, seal tightly and store at -20ºC.Store the other components at 0-5ºC.♦ Since reactive reagent binds to an amino group in antibody, there is a possibility that the labeled antibody loses the antigen recognition ability (Table 2).

Table. 1 Compatible concentrations of the additives

Table. 2 Non-Compatible Antibody

 

Fig. 3 Microscope Image of Mitochondria in Hela CellsPeroxidase were to anti-mitochondria antibody using Ab-10 Rapid Peroxidase Labeling Kit (code：LK33)