日期：2022年4月30日

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%.