肿瘤成像－IRDye800CW RGD Optical Probe
该探针由IRDye800CW的染料标记的环状五肽组成，识别基团RGD是一个三肽序列(Arginine, Glycine and Aspartic Acid)。RGD基团能特异结合细胞表面受体αvβ3 （细胞表面的异二聚体糖蛋白），可以研究和监测αvβ3受体过表达的相关疾病。该光学探针已经被证明能特异结合许多肿瘤细胞系。如U87, A431, PC3M-LN4和 22Rv1。
Data Courtesy of Dr. Kai Chen, StanfordUniversity
Intercept (PBS) Blocking Buffers are formulated to provide highly efficient blocking and low background variability for replicable quantitative Western blots and other immunoassays. Intercept (PBS) Blocking Buffer is the blocking buffer of choice for near-infrared fluorescent applications when using a phosphate-buffered saline (PBS) system.
Contains no mammalian proteins for the lowest cross-reactivity with mammalian antibodies.
Excellent choice for biotin-streptavidin detection.
Suitable for a wide range of applications, including Western blots, In-Cell Western™ assays, and protein arrays.
For larger pack sizes, please contact your local sales office.
Intercept (PBS) Blocking Buffer is a ready-to-use blocker formulation in PBS. It contains no sodium azide, making it suitable for both near-infrared or chemiluminescent detection. It is stored at 4 °C and is also available in kits with Immobilon®-FL PVDF and nitrocellulose membranes.
Figure 1: Western blot protocol demonstrating the comparison of Intercept Blocking Buffer to Odyssey Blocking Buffer. Blots with serial dilution of mouse heart tissue extract were probed for Stat3 Mouse mAb and p38 MAPK Rabbit Ab, detected with IRDye® 800CW Goat anti-Mouse IgG (P/N 925-32210) and IRDye 680LT Goat anti-Rabbit IgG (P/N 925-68021), and scanned together on an Odyssey CLx Imaging System.图 1：Western 印迹协议，展示了 Intercept Blocking Buffer 与 Odyssey Blocking Buffer 的比较。 使用 IRDye® 800CW 山羊抗小鼠 IgG（部件号 925-32210）和 IRDye 680LT 山羊抗兔 IgG（部件号/ N 925-68021)，并在 Odyssey CLx 成像系统上一起扫描。
Intercept (PBS) Blocking Buffer can be used for many immunoassays and applications, including:
Intercept (TBS) Blocking Buffers are formulated to provide highly efficient blocking and low background variability for replicable quantitative Western blots and other immunoassays. Intercept (TBS) Blocking Buffer is a ready-to-use blocker formulation in Tris-buffered saline (TBS).
Contains no mammalian proteins for the lowest cross-reactivity with mammalian antibodies.
Can be used with the widest range of targets for Western blot detection.
Ideal for primary antibodies that are optimized for Tris-based buffer systems.
Can be used for detection of phosphorylated targets.
Has superior performance for alkaline phosphatase detection.
Excellent choice for biotin-streptavidin detection.
For larger pack sizes, please contact your local sales office.
Intercept (TBS) 封闭缓冲液经过配制，可为可复制的定量蛋白质印迹和其他免疫测定提供高效封闭和低背景变异性。 Intercept (TBS) 封闭缓冲液是一种在 Tris 缓冲盐水 (TBS) 中的即用型封闭剂配方。
不含哺乳动物蛋白，与哺乳动物抗体的交叉反应性最低。
可用于最广泛的蛋白质印迹检测目标。
非常适合针对基于 Tris 的缓冲系统进行优化的一抗。
可用于检测磷酸化目标。
具有卓越的碱性磷酸酶检测性能。
生物素-链霉亲和素检测的绝佳选择。
如需更大的包装尺寸，请联系您当地的销售办事处。
Formulation
Intercept (TBS) Blocking Buffer is a ready-to-use blocker formulation in TBS. It contains no sodium azide, making it suitable for both near-infrared or chemiluminescent detection. It is stored at 4 °C and is also available in kits with Immobilon®-FL PVDF and nitrocellulose membranes.
Figure 1: Western blot protocol demonstrating the comparison of Intercept Blocking Buffer to Odyssey Blocking Buffer. Blots with serial dilution of mouse heart tissue extract were probed for Stat3 Mouse mAb and p38 MAPK Rabbit Ab, detected with IRDye® 800CW Goat anti-Mouse IgG (P/N 925-32210) and IRDye 680LT Goat anti-Rabbit IgG (P/N 925-68021), and scanned together on an Odyssey CLx Imaging System.图 1：Western 印迹协议，展示了 Intercept Blocking Buffer 与 Odyssey Blocking Buffer 的比较。 使用 IRDye® 800CW 山羊抗小鼠 IgG（部件号 925-32210）和 IRDye 680LT 山羊抗兔 IgG（部件号/ N 925-68021)，并在 Odyssey CLx 成像系统上一起扫描。
Intercept (TBS) Blocking Buffer can be used for many immunoassays and applications, including:
Intercept (PBS) Protein-Free Blocking Buffers are formulated to provide highly efficient blocking and low background variability for replicable quantitative Western blots and other immunoassays. This buffer contains no proteins, for assays where use of animal-based products is prohibited.
Excellent choice for biotin-streptavidin detection.
Suitable for a wide range of applications, including Western blots, In-Cell Western™ assays, and protein arrays.
For larger pack sizes, please contact your local sales office.
Intercept (PBS) Protein-Free Blocking Buffer is a ready-to-use blocker formulation in phosphate-buffered saline and is stored at 4 °C. It contains no sodium azide, making it suitable for both near-infrared or chemiluminescent detection.
Intercept (PBS) 无蛋白封闭缓冲液是一种在磷酸盐缓冲盐水中的即用型封闭剂配方，可在 4 °C 下储存。 它不含叠氮化钠，因此适用于近红外或化学发光检测。
Shake well before each use.
Intercept (PBS) Protein-Free Blocking Buffer can be used for many immunoassays and applications, including:
Intercept T20 Antibody Diluents for use with Odyssey® Imaging Systems, are optimized for use with IRDye® Infrared Dye Reagents and other near-infrared fluorophores. All Intercept T20 Antibody Diluents provide excellent performance for quantitative and chemiluminescent Western blots, as well as other immunoassays.
Antibody diluents are used for the dilution of primary antibodies and secondary antibodies to the working concentration required for your experiments. Antibody diluents improve the specificity of primary and secondary antibodies, reducing off-target effects. This reduces background due to non-specific binding.
Intercept T20 Antibody Diluents are preformulated in-house with Tween® 20, a non-ionic detergent. There’s no need to mix the diluent yourself, which saves you time and reduces potential variation.
Intercept (TBS) Protein-Free Blocking Buffers are formulated to provide highly efficient blocking and low background variability for replicable quantitative Western blots and other immunoassays where animal-based products are prohibited. Intercept (TBS) Protein-Free Blocking Buffer is a ready-to-use blocker formulation in Tris-buffered saline (TBS).
Intercept (TBS) 无蛋白封闭缓冲液经过配制，可为可复制的定量蛋白质印迹和其他禁止使用动物产品的免疫分析提供高效封闭和低背景变异性。 Intercept (TBS) 无蛋白封闭缓冲液是一种在 Tris 缓冲盐水 (TBS) 中的即用型封闭剂配方。
Contains no proteins, for assays where animal-based products are prohibited.
Can be used with the widest range of targets for Western blot detection.
Ideal for primary antibodies that are optimized for Tris-based buffer systems.
Can be used for detection of phosphorylated targets.
Has superior performance for alkaline phosphatase detection.
Excellent choice for biotin-streptavidin detection.
不含蛋白质，用于禁止动物产品的化验。
可用于最广泛的蛋白质印迹检测目标。
非常适合针对基于 Tris 的缓冲系统进行优化的一抗。
可用于检测磷酸化目标。
具有卓越的碱性磷酸酶检测性能。
生物素-链霉亲和素检测的绝佳选择。
For larger pack sizes, please contact your local sales office.
Formulation
Intercept (TBS) Protein-Free Blocking Buffer is a ready-to-use blocker formulation in TBS and is stored at 4 °C. It contains no sodium azide, making it suitable for both near-infrared or chemiluminescent detection.
Intercept T20 (PBS) Protein-Free Antibody Diluent improves the specificity of the primary and secondary antibodies, reducing off-target effects. The diluent contains Intercept (PBS) Protein-Free Blocking Buffer preformulated with Tween® 20 for use in a phosphate-buffered saline (PBS) system. This diluent can be used where animal-based products are prohibited.
Since there’s no need to mix the diluent yourself, it saves you time and reduces potential variation.
Intercept T20 (TBS) Antibody Diluent improves the specificity of the primary and secondary antibodies, reducing off-target effects. The diluent contains Intercept (TBS) Blocking Buffer in Tris-buffered saline (TBS) preformulated with Tween® 20.
Since there’s no need to mix the diluent yourself, it saves you time and reduces potential variation.
Intercept® (TBS) Blocking Buffer and Diluent Kit,Intercept® (TBS) 封闭缓冲液和稀释剂试剂盒
Intercept® (PBS) Blocking Buffer and Diluent Kit,Intercept® (PBS) 封闭缓冲液和稀释剂试剂盒
Figure 1: Western blot protocol demonstrating the comparison of Intercept Blocking Buffer to Odyssey Blocking Buffer. Blots with serial dilution of mouse heart tissue extract were probed for Stat3 Mouse mAb and p38 MAPK Rabbit Ab, detected with IRDye 800CW Goat anti-Mouse IgG and IRDye 680LT Goat anti-Rabbit IgG, and scanned together on an Odyssey CLx Imaging System.
Intercept T20 (TBS) Protein-Free Antibody Diluent improves the specificity of the primary and secondary antibodies, reducing off-target effects. The diluent contains Intercept (TBS) Protein-Free Blocking Buffer in Tris-buffered saline (TBS) preformulated with Tween® 20. This diluent can be used where animal-based products are prohibited.
Since there’s no need to mix the diluent yourself, it saves you time and reduces potential variation.
Diluent C for Use with CellVue® Fluorescent Cell Labeling Kits,用于 CellVue® 荧光细胞标记试剂盒的稀释剂 C
Fluorescently Label Cells or Tissues for in vivo or in vitro Studies
CellVue fluorescent imaging kits use proprietary labeling technology to stably incorporate fluorescent dyes containing long aliphatic hydrocarbon tails into lipid membranes1. They are useful for researchers working in all aspects of science and technology where fluorescently-labeled cells and/or tissues are required. CellVue dyes also provide researchers with valuable tools for many in vivo and in vitro cell studies using fluorescent membrane labels.
The CellVue dyes are provided in an easy-to-use kit format containing a solution of the dye in ethanol and a cell labeling diluent. The labeling diluent, Diluent C, provided with the kit is an iso-osmotic aqueous solution that contains no physiologic salts or buffers, detergents, or organic solvents, and is designed to maintain cell viability while maximizing dye solubility and staining efficiency2, 3. The simple membrane labeling protocol provided with the kit is applicable to almost any type of cell or organism.
CellVue 染料以易于使用的试剂盒形式提供，其中包含染料乙醇溶液和细胞标记稀释剂。 试剂盒随附的标记稀释剂 Diluent C 是一种等渗水溶液，不含生理盐或缓冲液、去污剂或有机溶剂，旨在保持细胞活力，同时最大限度地提高染料溶解度和染色效率 2, 3。 试剂盒提供的简单膜标记方案适用于几乎任何类型的细胞或生物体。
Molecular Structure of CellVue Dyes
CellVue dyes consist of long aliphatic hydrocarbon tails linked to a polar fluorescent chromophore.
These extremely lipophilic fluorescent dyes rapidly and stably integrate into the phospholipid membrane of cells or other membrane-containing bioparticles by non-covalent interactions.
The dyes are stably maintained within the lipid bilayer through strong hydrophobic interactions and do not transfer into the unstained membranes of adjacent cells, which permits a labeled cell to be tracked for extended periods of time.
The rapid incorporation of the dye allows for immediate analysis of cell functions without a waiting period.
Horan, P. K., and Slezak, S. E., Nature, 340, 167-168 (1989).
Horan, P. K., et al., Methods Cell Biol., 33, 469-490 (1990).
Poon, R.Y., et al. In Living Color: Flow Cytometry and Cell Sorting Protocols, Diamond, R. A., and DeMaggio, S. (Eds.). p. 302-352 (Springer-Verlag, New York, 2000).
10X Orange Loading Dye is used as a loading buffer for DNA gel electrophoresis. This loading dye is not visualized when imaged on Odyssey® Imagers. The primary application for 10X Orange Loading Dye is for electrophoretic mobility shift assays (EMSA). When run on a 5% native acrylamide 1X TBE gel, the orange dye migrates with the 20-28 bp DNA fragment.
10X Orange Loading Dye 用作 DNA 凝胶电泳的上样缓冲液。 当在 Odyssey® Imagers 上成像时，这种加载染料不可见。 10X Orange Loading Dye 的主要应用是电泳迁移率变动分析 (EMSA)。 在 5% 天然丙烯酰胺 1X TBE 凝胶上运行时，橙色染料与 20-28 bp DNA 片段一起迁移。
Use Near-Infrared Click Chemistry Reagents for Biomolecule Labeling
Biomolecule labeling continues to be a cornerstone feature of many in vitro and in vivo biological experiments. Click Chemistry has become convenient and reliable method for labeling a wide variety of molecules for applications ranging from biomarker isolation to assay development.
Click Chemistry reagents from LI-COR offer several benefits:
Versatile alternatives to affinity-based detection
Chemoselectivity and easy-to-perform, high yielding reactions
Flexibility with a choice for copper-catalyzed and copper-free reactions
Click Chemistry Reactions
Click Chemistry utilizes pairs of reagents that exclusively react with each other and are effectively inert to naturally occurring functional groups such as amines.
Click Chemistry reactions can be categorized into two separate groups, copper-catalyzed or copper-free. Copper-catalyzed Click Chemistry is used for initiating reactions between azides and alkynes. Although they initiate and accelerate Click Reactions, copper catalysts are cytotoxic and inappropriate for use in living systems.
To address this limitation, copper-free methods have been developed to allow Click Chemistry with live cells and whole organisms.
Reagents > 4X Protein Sample Loading Buffer for Western Blots
4X Protein Sample Loading Buffer is optimized for use as a loading buffer for protein gel electrophoresis. This orange loading buffer is recommended for use with Odyssey® Imaging Systems as it does not fluoresce in the 700 nm channel the way blue loading buffers do. The buffer is optimized for use with SDS-PAGE and Tris-Glycine-SDS running buffer.
Mobility shift assay protocols can be easily converted to infrared fluorescent assays by replacing the existing DNA oligonucleotides with IRDye® infrared dye end-labeled oligonucleotides. Binding and electrophoresis conditions are the same as any other EMSA detection method.
A DNA oligonucleotide end-labeled with IRDye 700 infrared dye is a good substrate for protein binding. Using the Odyssey® Infrared Imaging System, IRDye infrared dye labeled DNA detection is linear within a 50-fold dilution range from 9.1 fmol to 0.18 fmol.
通过用 IRDye® 红外染料末端标记的寡核苷酸替换现有的 DNA 寡核苷酸，迁移率变化分析方案可以轻松转换为红外荧光分析。 结合和电泳条件与任何其他 EMSA 检测方法相同。
用 IRDye 700 红外染料末端标记的 DNA 寡核苷酸是蛋白质结合的良好底物。 使用 Odyssey® 红外成像系统，IRDye 红外染料标记的 DNA 检测在 9.1 fmol 至 0.18 fmol 的 50 倍稀释范围内是线性的。
Perform NIR Fluorescent EMSA and Save Time
Infrared fluorescent assays can be completed in less than two hours with no gel transfer or film exposure. The gel doesn’t even have to be removed from the glass plates for imaging. If you are not satisfied that the electrophoresis has progressed far enough, you can place the gel back into the electrophoresis unit and run longer.
Other IRDye 700 oligonucleotides as well as IRDye 800 oligonucleotides are available through Integrated DNA Technologies, TriLink BioTechnologies, or Metabion International AG.
其他 IRDye 700 寡核苷酸和 IRDye 800 寡核苷酸可通过 Integrated DNA Technologies、TriLink BioTechnologies 或 Metabion International AG 获得。
Near-Infrared Fluorescence Detection for Gel Shift Assays Has Advantages over Use of Radioisotopes
Infrared
Radioisotope
Easy access and disposal
Short half-life of the label
Dye is stable for a long time
Regulatory procedures
Disposal limitations
Non-hazardous
Hazardous
Gel (glass plates) can be easily imaged on the Odyssey
Reagents > 4X Protein Sample Loading Buffer and PVDF Membrane Kit
4X Protein Sample Loading Buffer (P/N 928-40004) is optimized for use as a loading buffer for protein gel electrophoresis. This orange-colored loading buffer has no dyes that may cause background as compare to other loading buffers with a blue dye component that can increase background.
The Immobilon®-FL PVDF membrane included in these kits has been tested to ensure a low-background for near-infrared fluorescent Western blot success.
Perform Fast, Cost-Effective Cell-Based Western Assays
The In-Cell Western Assay is an immunocytochemical assay that uses fluorescence to detect and quantify proteins in cells that have been cultured in microplates. Detecting proteins in their cellular context increases quantification precision.
This process allows for higher throughput compared to Western blotting and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. Cost per well is reduced to a fraction of the cost of typical screening methods with CellTag™ Stain In-Cell Western Kits.
CellTag Stains are a fluorescent, non-specific cell stain that provides accurate normalization to cell number for In-Cell Western applications. The stain accumulates in both the nucleus and cytoplasm of permeabilized cells and provides linear fluorescent signal across a wide range of cell types and cell numbers. CellTag Stains are applied to the cells during incubation with IRDye® Secondary Antibodies and enable accurate measurement of target protein levels with much higher throughput than Western blotting.
In-Cell Western 检测是一种免疫细胞化学检测，它使用荧光检测和量化微孔板中培养的细胞中的蛋白质。在细胞环境中检测蛋白质可提高定量精度。
与蛋白质印迹法相比，该过程允许更高的通量，并消除了典型的蛋白质印迹法步骤，例如细胞裂解物制备、电泳和膜转移。使用 CellTag™ Stain In-Cell Western Kits 将每孔成本降低到典型筛选方法成本的一小部分。
CellTag 染色剂是一种荧光非特异性细胞染色剂，可为 In-Cell Western 应用提供准确的细胞数标准化。该染料在透化细胞的细胞核和细胞质中积累，并在各种细胞类型和细胞数量中提供线性荧光信号。在与 IRDye® 二抗孵育期间将 CellTag Stains 应用于细胞，并能够以比蛋白质印迹高得多的通量准确测量靶蛋白水平。
IRDye Infrared Fluorescent Dyes and near-infrared (NIR) fluorescence imaging deliver enhanced sensitivity due to low background autofluorescence in the near-infrared region and, therefore, higher signal-to-noise ratios.
IRDye Fluorescent Dyes have absorption and emission wavelengths in the NIR spectrum, between 680 and 800 nm. Most of the IRDye reagents are compatible with the Odyssey® DLx, Odyssey XF, Odyssey Sa, Pearl® Imagers, and 4300 DNA Analyzer platforms.
Standard NHS ester chemistry is used to produce custom probes labeled with LI-COR IRDye Infrared Dyes. The NHS ester reactive group provides the functionality for labeling primary and secondary amino groups.
Maleimides
IRDye maleimides provide the functionality for labeling molecules that contain free sulfhydryl (-SH) groups with IRDye Infrared Dyes. The reactive group of the IRDye maleimide allows conjugation reactions to be performed very efficiently at physiological pH.
Assays that use infrared dye conjugates (such as in vivo imaging and cell binding assays) may require a “dye-only” control for potential effects or retention of the dye. The carboxylate dye has no reactive group and cannot be used for labeling.
Carboxylate dye forms can serve as an ideal control for cell-based assays that monitor binding of a dye-labeled agent to validate optical agents for in vivo administration and to evaluate binding specificity. This dye form can also be used to evaluate the behavior and clearance of the dye itself and as a standard to determine the amount of unreacted (“free”) dye after conjugation and purification.
IRDye Protein Labeling Kits label antibodies, other proteins, and peptides for applications such as Western blots, In Cell Western™ assays, in vivo imaging, and whole organ or tissue section assays. Applications of dye-labeled conjugates vary, depending on the IRDye used for labeling.
IRDye Labeling Kits
DBCO, Alkyne, and Azide Dye Forms Used for Click Chemistry
Biomolecule labeling continues to be a cornerstone feature of many in vitro and in vivo biological experiments. Click Chemistry has recently emerged as a convenient, versatile, and reliable method for labeling a wide variety of molecules for applications ranging from biomarker isolation to assay development. Click Chemistry utilizes pairs of reagents that exclusively react with each other and are effectively inert to naturally occurring functional groups such as amines
LI-COR offers DBCO, azide, and alkyne dye forms of IRDye 800CW and IRDye 680RD.
Click Chemistry Dyes
IRDye Properties and Forms Available
Dye
Exmax (nm)*
Emmax (nm)*
NHS Ester
Maleimide
Carboxylate
DBCO
Azide
Alkyne
Phosphoramidite
LI-COR Channel
IRDye 800CW
778
794
800 nm
IRDye 800RS
770
786
800 nm
IRDye 800 phosphoramidite
787
812
800 nm
IRDye 750
766
776
IRDye 700 phosphoramidite
680
697
700 nm
IRDye 680LT**
680
694
700 nm
IRDye 680RD
680
694
700 nm
* Data collected using methanol as the solvent.
** IRDye 680LT dye products should not be used for small animal in vivo imaging or In-Cell Western Assays. The higher level of fluorescent intensity creates high background making it unfavorable for use in these applications. We recommend using IRDye 680RD dye products for these applications.
Which 700 Channel Dye Should I Use?
LI-COR offers three distinct 700 channel dyes for a variety of applications. Choosing the correct dye is critical when developing an experiment. A summary of each dye and an application table are provided to help you determine which dye is best for your needs.
IRDye 680RD
IRDye 680RD is the near-infrared fluorescent dye of choice for small animal imaging, Western blot, and In-Cell Western Assay applications in the 700nm channel. It has the lowest background compared to LI-COR’s other 700 nm dyes and can be used for the widest variety of applications with little optimization. The NHS ester and maleimide forms of this infrared dye are ideal for labeling proteins, peptides, and antibodies. LI-COR offers a variety of IRDye 680RD dyes, labeling kits and secondary antibodies.
IRDye 680LT
IRDye 680LT is significantly brighter and more photostable than many other 700 nm near-infrared dyes such as Alexa Fluor® 680. It provides superior performance for protein detection applications, including microscopy and Western blot applications. LI-COR offers a variety of IRDye 680LT dyes, labeling kits, and secondary antibodies. When using IRDye 680LT secondary antibodies for Western blot detection, it is critical to follow the recommendations in the pack insert. Dilution optimization may be required to achieve best results.
Application
IRDye 680RD
IRDye 680LT
Western Blot
In-Cell Western Assay
not recommended
On-Cell Western Assay
not recommended
RNAi Screens
for Western blots only
Protein Array
Immunohistochemistry
Microscopy
2D Gel Detection
Tissue Section Imaging
FRET-based Assays
Small Animal Imaging
not recommended
IRDye Infrared Dyes are Optimized for Near-Infrared Fluorescent Detection
IRDye Near-Infrared Dyes are optimized for excitation and detection in the near-infrared spectrum, providing numerous benefits.
Low background autofluorescence allows higher signal to noise ratios (see Figure 1)
Wide range of dyes and functional groups available for multiple applications
Excellent photostability
Dye brightness
Figure 1. Animal tissue absorbs visible light. Hemoglobin (Hb) and other tissue components strongly absorb visible light. In the NIR region, where infrared agents are detected, tissue absorbance is dramatically reduced. Above 820 nm, light absorbance by water begins to increase.
LI-COR offers IRDye 800CW that has been manufactured under current good manufacturing practices (cGMP) and is available for conjugation to targeted biomolecules for investigational use in clinical trials. In September of 2007, LI-COR Biosciences announced the successful completion of animal toxicity studies of its IRDye 800CW infrared dye carboxylic acid using a protocol reviewed by the Food and Drug Administration. The completion of the toxicity studies was a key milestone in the development of the IRDye 800CW dye for potential clinical imaging use. The complete report was published in 2010 and is available to the public. In addition, drug master files (DMF) are on record with U.S. and European regulatory authorities.
LI-COR IRDye Infrared Fluorescent Dyes are used for protein and cellular assays, microscopy, and in vivo molecular imaging in animals for research purposes. IRDye 800CW is cited in a wide variety of published non-clinical research for labeling nucleic acids, antibodies, proteins, and peptides where there is a need for high signal, low background imaging. With an 800nm emission wavelength, the IRDye 800CW dye is spectrally ideal for animal imaging research.
While the IRDye 800CW dye has successfully completed toxicity studies, it has not been studied for diagnostic or therapeutic use in humans, and has not been approved by the Food and Drug Administration for this use. LI-COR is exploring options for additional studies as next steps in the Food and Drug Administration approval process leading toward clinical use.
LI-COR IRDye Infrared Dyes are Being Used in Clinical Trials
IRDye 800CW conjugated probes and agents have propelled more than 20 Phase I and Phase II clinical trials, To learn more about LI-COR infrared dye conjugates being used in clinical trials, visit Probe Development Clinical Applications.
To discuss purchase quantities over 1 gram or commercial development rights using IRDye infrared dyes, please contact Business Development.
Reagents > 96-Well Plate for In-Cell Western™ Assays
Sterile cell culture plates optimized for use for the In-Cell Western Assay. These plates include a lid and have a clear, polystyrene bottom with black walls.
无菌细胞培养板优化用于 In-Cell Western Assay。 这些盘子包括一个盖子，并有一个透明的聚苯乙烯底部和黑色的墙壁。
IRDye® 800CW Protein Labeling Kits,IRDye® 800CW 蛋白质标记试剂盒
IRDye® 680RD Protein Labeling Kits,IRDye® 680RD 蛋白质标记试剂盒
IRDye® 680LT Protein Labeling Kits,IRDye® 680LT 蛋白质标记试剂盒
IRDye® 650 Protein Labeling Kits,IRDye® 650 蛋白质标记试剂盒
IRDye® Protein Labeling Kits label antibodies and other proteins for applications such as Western blots, In Cell Western™ assays, in vivo imaging, and whole organ or tissue section assays. Applications vary depending on the IRDye infrared dye selected for labeling. Please refer to specific dye page to find out which applications apply to each IRDye NHS ester.
IRDye reactive dyes bear an N-hydroxysuccinimide (NHS) reactive group that couples to free amino groups and forms a stable conjugate. Proteins other than IgG antibodies can be labeled, but protocol adjustment and optimization may be necessary.
IRDye labeling kits are based on a simple conjugation protocol that uses fast, spin column cleanup with resulting purity equivalent to purification by dialysis. Simply dissolve the protein in the provided buffer, combine it with the appropriate amount of water-soluble amine-reactive IRDye, and separate the labeled conjugate from the free dye using the included spin columns. Labeling and purification are complete in approximately 2 hours.
For more information, refer to the IRDye Peptide Labeling Application Guide.
Streptavidin binds to biotin very strongly and is used to detect biotinylated proteins.
Fluorescent dye-labeled streptavidins can be used as a secondary detection reagent for microscopy, protein arrays, In-Gel Westerns, and Western blotting.
All streptavidins are supplied as a liquid in buffer containing 10 mM phosphate, 183 mM NaCl, 2.7 nM KCl, pH 7.4 with sodium azide 0.005% (w/v) as a preservative.
To use, centrifuge briefly before use to eliminate aggregates that may have formed in solution. For IRDye® Streptavidin, a final concentration of 0.2 to 1.0 µg/ml (1:1,000 to 1:5,000) is usually satisfactory for most applications. For VRDye™ Streptavidin, a concentration of 1:2,000 to 1:5,000 is usually satisfactory for Western blotting. However, appropriate dilution may need to be determined empirically.
For membrane-based applications and In-Gel Westerns, it is recommended to add SDS (0.02% to 0.1% final concentration), in addition to Tween® 20 (0.1 to 0.2% final concentration) during the detection incubation step to reduce non-specific background staining.
所有链霉亲和素均以缓冲液形式提供，缓冲液中含有 10 mM 磷酸盐、183 mM NaCl、2.7 nM KCl，pH 7.4，叠氮化钠 0.005% (w/v) 作为防腐剂。
Figure 1. Example of IRDye Streptavidin used for a protein array. Spots are Biotinylated BSA + 2-fold serial dilution of IRDye 680 Streptavidin. Data were obtained from a 21-micron scan on an Odyssey Infrared Imaging System.图 1. 用于蛋白质阵列的 IRDye 链霉亲和素示例。 斑点是生物素化的 BSA + IRDye 680 Streptavidin 的 2 倍连续稀释。 数据来自 Odyssey 红外成像系统上的 21 微米扫描。
Reagents > CellTag™ 520 Stain for In-Cell Western™ Assays
CellTag 520 Stain is a visible fluorescent, non-specific cell stain that provides accurate normalization to cell number for In-Cell Western™ Assay applications.
The stain accumulates in both the nucleus and cytoplasm of permeabilized cells, and provides linear fluorescent signal across a wide range of cell types and cell numbers.
CellTag 520 Stain is detected in the 520 nm channel. CellTag 520 Stain is applied to the cells during incubation with an IRDye® Secondary Antibodies (e.g., IRDye 680RD or IRDye 800CW), and enables accurate measurement of target protein levels with much higher throughput than Western blotting.
Linear Relationship Between Fluorescence and Cell Number
Two-fold serial dilutions of NIH3T3 cells were plated in a 96-well plate. Cells were fixed, permeabilized, stained with CellTag 520 Stain (0.1 μM), and detected with the Odyssey M Imaging System.
PBS Blocking Buffer and PVDF Kits,PBS 封闭缓冲液和 PVDF 试剂盒
TBS Blocking Buffer and PVDF Kits,TBS 封闭缓冲液和 PVDF 试剂盒
Odyssey® Nitrocellulose Membranes,Odyssey® 硝酸纤维素膜
4X Protein Sample Loading Buffer and PVDF Membrane Kit,4X 蛋白质上样缓冲液和 PVDF 膜试剂盒
A low-background membrane is essential for infrared Western blot success. Background can be attributed to membrane autofluorescence or to detection of antibody non-specifically binding to the membrane.
低背景膜对于红外蛋白质印迹的成功至关重要。 背景可归因于膜自发荧光或检测到与膜非特异性结合的抗体。
Rely on Quality and Performance with LI-COR Western Blot Membranes
LI-COR has evaluated many different membranes for Western blotting; examples of membrane performance can be seen in the figure below. There is typically more variability in PVDF membrane performance than for nitrocellulose membranes.
LI-COR offers high-quality, prescreened Immobilon®-FL PVDF, without lot-to-lot variability, packaged in affordable kits with the Intercept™ Blocking Buffer (PBS or TBS), or with 4X Protein Sample Loading Buffer. LI-COR also offers nitrocellulose membranes in both sheets and rolls.
0.45 µm; 26.5 cm × 3.75 m roll + 500 mL Intercept (PBS) Blocking Buffer
TBS Blocking Buffer (500 mL) and PVDF Kit
926-32099
0.45 µm; 26.5 cm × 3.75 m roll + 500 mL Intercept (TBS) Blocking Buffer
4X Protein Sample Loading Buffer and PVDF Membrane Kit
926-31097
0.45 µm; 26.5 cm × 3.75 m roll + 15 mL 4X Protein Sample Loading Buffer
PBS Blocking Buffer (10 x 500 mL) and PVDF Kit
829-32080
0.45 µm; 26.5 cm × 3.75 m roll + 10 × 500 mL Intercept (PBS) Blocking Buffer
TBS Blocking Buffer (10 x 500 mL) and PVDF Kit
926-32100
0.45 µm; 26.5 cm × 3.75 m roll + 10 × 500 mL Intercept (TBS) Blocking Buffer
Western Blot Membrane Selection Can Affect Your Results
Western blot detection of transferrin using various vendors and brands of PVDF membrane. PVDF membranes were scanned on the Odyssey Classic Infrared Imaging System in both 700 and 800 nm channels. LI-COR offers high-quality, prescreened Immobilon-FL PVDF membranes in convenient, affordable kits.使用各种供应商和品牌的 PVDF 膜对转铁蛋白进行蛋白质印迹检测。 在 Odyssey Classic Infrared Imaging System 上以 700 和 800 nm 通道扫描 PVDF 膜。 LI-COR 在方便、经济的套件中提供高质量、预筛选的 Immobilon-FL PVDF 膜。
Reagents > CellTag™ 700 Stain for In-Cell Western™ Assays
CellTag 700 Stain is a near-infrared fluorescent, non-specific cell stain that provides accurate normalization to cell number for In-Cell Western™ Assay applications.
The stain accumulates in both the nucleus and cytoplasm of permeabilized cells, and provides linear fluorescent signal across a wide range of cell types and cell numbers.
CellTag 700 Stain is detected in the 700 nm channel of Odyssey® CLx, Classic, and Sa Imaging Systems. CellTag 700 Stain is applied to the cells during incubation with an IRDye® 800CW secondary antibody, and enables accurate measurement of target protein levels with much higher throughput than Western blotting.
Linear Relationship Between Fluorescence and Cell Number
Two-fold serial dilutions of A431 and NIH/3T3 cells were plated in 96-well plates. Cells were fixed, permeabilized, stained with CellTag 700 Stain (0.2 μM), and detected with the Odyssey Classic Infrared Imaging System. Trim Signal values were used to generate the graphs.将 A431 和 NIH/3T3 细胞的两倍系列稀释液接种在 96 孔板中。 细胞被固定、透化、用 CellTag 700 Stain (0.2 μM) 染色，并用 Odyssey Classic 红外成像系统检测。 修剪信号值用于生成图表。
Evaluate Sample Loading Consistency and Uniformity with Odyssey Loading Indicators
Odyssey Loading Indicators (OLI) provide a simple, convenient method to evaluate the consistency of sample loading volume across gel lanes, as well as the uniformity of Western blot transfer.
It is essential to have consistent sample loading between gel lanes for comparative and quantitative Western blot analysis. By adding equal amounts of an Odyssey Loading Indicator, you can determine if loading was consistent or if a pipetting error occurred.
Evaluate Consistency. Ensure samples were equally loaded in each lane.
Check Uniform Transfer. Confirm that transfer from gel to membrane was uniform.
Assess Stable Expression. Help determine if a housekeeping protein is stably expressed and can be used for Western blot normalization.
Note: Accurate normalization relies on an internal loading control, an endogenous protein used as an indicator of sample concentration. Since the OLI is an exogenous protein, it functions as an external loading control, and cannot be used for normalization. However, you can use OLI to help check if your internal loading control is stably expressed.
Visit our Western blot Normalization page for more information on Western blot normalization strategies.
Assess Stable Expression of Housekeeping Proteins for Normalization
If you’re using a housekeeping protein to normalize Western blot data, you must validate that its expression is constant across all samples and is unaffected by experimental conditions. As an exogenous protein, the Odyssey Loading Indicator is not subject to up- or down-regulation as a function of experimental sample treatment. A stable signal for an OLI across several lanes indicates that any signal variation for the housekeeping protein was not caused by inconsistent sample loading.
如果您使用看家蛋白来标准化蛋白质印迹数据，您必须验证其在所有样品中的表达是恒定的，并且不受实验条件的影响。 作为外源性蛋白质，Odyssey Loading Indicator 不受实验样品处理的上调或下调影响。 跨多个泳道的 OLI 信号稳定表明管家蛋白的任何信号变化都不是由不一致的样品加载引起的。
“‘House-keeping’ proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression.”
Figure 2. Assessing stable expression of tubulin and COX IV with OLI. The housekeeping protein signal intensity was plotted, along with signal intensities of OLI and Revert 700 Total Protein Stain. Both (A) tubulin and (B) COX IV signals varied in response to treatment, while Revert 700 Total Protein Stain signals and OLI signals remained constant. These quantitative data indicate the expression of both housekeeping proteins was affected by Etoposide treatment.
图 2. 使用 OLI 评估微管蛋白和 COX IV 的稳定表达。 绘制管家蛋白信号强度以及 OLI 和 Revert 700 总蛋白染色剂的信号强度。 (A) 微管蛋白和 (B) COX IV 信号均随治疗而变化，而 Revert 700 Total Protein Stain 信号和 OLI 信号保持不变。 这些定量数据表明两种看家蛋白的表达都受到依托泊苷处理的影响。
The CellVue Burgundy cell labeling kit uses proprietary membrane labeling technology to stably incorporate CellVue Burgundy, a fluorescent dye with long aliphatic tails, into lipid regions of the cell membrane1. The labeling buffer provided with the kit (Diluent C) is an iso-osmotic aqueous solution which contains no physiologic salts or buffers, detergents, or organic solvents and is designed to maintain cell viability while maximizing dye solubility and staining efficiency. The labeling efficiency of staining is dependent upon the cell type being labeled and the membranes of the cells2, 3.
Use NIR Fluorescent Labeling for Cell Tracking and Cell Proliferation Studies
CellVue products have been reported to be useful for short-term in vitro cell proliferation studies4, cell tracking in isolated organ preparation applications5, and cell tracking using the Pearl® Small Animal Imaging System or Odyssey® CLx or Odyssey Classic Infrared Imaging System6.
Kit Components
CellVue Burgundy dye stock (1 vial containing 0.1 mL, 1 x 10-3 M in ethanol)
Diluent C (1 vial containing 10 mL, sufficient for 5 labeling reactions)
CellVue 勃艮第染料储备（1 瓶含 0.1 mL，1 x 10-3 M 乙醇溶液）
稀释剂 C（1 瓶含有 10 mL，足以进行 5 次标记反应）
Excitation and Emission Properties of CellVue Burgundy Dye
The excitation and emission properties of CellVue Burgundy dye are compatible with a range of commercially available near-infrared fluorescent plate readers, flow cytometers, in vitro and in vivo fluorescent imaging systems, and confocal microscopes.
CellVue Burgundy Excitation and Emission Spectra (ex max = 683 nm;em max = 707 nm).
Data Example using CellVue Burgundy Kit
Two-color imaging of CellVue Burgundy and CellVue NIR815 stains with the Odyssey Imager. A monocyte cell line (U937) was labeled with CellVue Burgundy (red), and a T-cell line (SUPT1) was labeled with CellVue NIR815 (green). The standard labeling protocol was used (1 x 107 cells/mL, 2 μM dye for 2-5 min at 25 °C). Labeled cells were spotted on a glass surface (106 cells/spot), with the cell spots partially overlapping. Cell spots were imaged with the Odyssey Infrared Imager, in two distinct fluorescent channels. A. Monocytes labeled with CellVue Burgundy were detected in the 700-nm channel (red). B. T-cells labeled with CellVue NIR815 were detected in the 800-nm channel (green). C. Simultaneous imaging in both fluorescent channels clearly shows both cell populations (overlapping signals are shown in yellow). Signal bleed-through between the two channels was not observed. Images courtesy of S. van de Waarsenburg and D. Anderson, Burnet Institute, Melbourne, Australia.
References
Horan, P. K., and Slezak, S. E., Nature, 340, 167-168 (1989).
Horan, P. K., et al., Methods Cell Biol., 33, 469-490 (1990).
Poon, R.Y., et al. In Living Color: Flow Cytometry and Cell Sorting Protocols, Diamond, R. A., and DeMaggio, S. (Eds.). p. 302-352 (Springer-Verlag, New York, 2000).
Stewart, C. C., Woodring, M. L., Podniesinski, E. and Gray, B. D. Flow Cytometer in the Infrared: Inexpensive Modifications to a Commercial Instrument. Cytometry, Part A, 67A, #2, 104-111 (2005).
Al-Mehdi, A-B, et al. Increased Depth of Cellular Imaging in the Intact Lung Using Far Red and Near Infrared Fluorescent Probes. Int. J. Biomed. Imag., Vol 2006, Article ID 37470, p 1-7.
Thomas, D. L., et al., Experimental Manipulations of Afferent Immune Responses Influence Efferent Immune Responses to Brain Tumors. Cancer Immunol. Immunother. Sep, 57(9): 1323-33 (2008).
CellVue is a registered trademark of PTI Research, Inc, used under license. CellVue products are sold under license from PTI Research, Inc.. US Patent # 8,029,767B2.
Every LI-COR BrightSite IRDye in vivo imaging agent has been carefully validated with cultured cell assays, microscopy, in vivo imaging of animal models, and histology to ensure high affinity and specificity.
BrightSite IRDye Small Animal Imaging Agents are:
Ready-to-use probes that are simple to administer, allowing you to begin animal studies immediately
Sensitive, offering the high signal-to-noise advantages of near-infrared fluorescence which are especially important for small or deep targets
Cited in several hundred peer-reviewed publications so you can use them with confidence
Imaged with any small animal imaging equipment with appropriate 680 nm or 800 nm filter sets
Compatible with most small animal imaging systems, including instruments from LI-COR Biosciences (Pearl® and Odyssey®), PerkinElmer (Xenogen, Caliper, CRi, and VisEn), and Bruker (Carestream and Kodak)
IRDye 800CW absorption/emission near 800 nm matches near-infrared absorption minima for bodily fluids and tissues, resulting in excellent tissue penetration, making it ideal for in vivo imaging.
Molar extinction coefficient characteristics of water, hemoglobin and oxygenated hemoglobin (400-1000 nm). IRDye infrared dyes have ideal excitation/emission wavelengths for in vivo imaging.
Detect Apoptotic and Necrotic Cells, Bacteria and Other Anionic Membranes
LI-COR also offers the PSVue® 794 Reagent Kit which can be used to detect apoptotic and necrotic cells, bacteria and other anionic membranes.
LI-COR 还提供 PSVue® 794 试剂盒，可用于检测凋亡和坏死细胞、细菌和其他阴离子膜。
Fluorescently Label Cells or Tissues for in vivo or in vitro Studies
CellVue® fluorescent imaging kits use proprietary labeling technology to stably incorporate fluorescent dyes containing long aliphatic hydrocarbon tails into lipid membranes.1 They are useful for researchers working in all aspects of science and technology where fluorescently-labeled cells and/or tissues are required. CellVue dyes also provide researchers with valuable tools for many in vivo and in vitro cell studies using fluorescent membrane labels.
CellVue dyes consist of long aliphatic hydrocarbon tails linked to a polar fluorescent chromophore.
These extremely lipophilic fluorescent dyes rapidly and stably integrate into the phospholipid membrane of cells or other membrane-containing bioparticles by non-covalent interactions
The dyes are stably maintained within the lipid bilayer through strong hydrophobic interactions and do not transfer into the unstained membranes of adjacent cells, which permits a labeled cell to be tracked for extended periods of time
The rapid incorporation of the dye allows for immediate analysis of cell functions without a waiting period
Molar extinction coefficient characteristics of water, hemoglobin and oxygenated hemoglobin (400-1000 nm). IRDye infrared dyes have ideal excitation/emission wavelengths for in vivo imaging.
Selecting the Right Optical Imaging Agent
Application
IRDye EGF
IRDye RGD
IRDye 2-DG
IRDye PEG
IRDye BoneTag
CellVue
PSVue
Tumor Imaging
Metabolic Imaging
Inflammation/Arthritis
Vasculature (Contrast)
Lymphatic Imaging
Lymph Node Imaging
Structural Imaging
Cell Trafficking
Apoptosis
References
Horan, P. K., and Slezak, S. E. (1989) Nature, 340. 167-168.
The CellVue NIR815 cell labeling kit uses proprietary membrane labeling technology to stably incorporate CellVue NIR815, a fluorescent dye with long aliphatic tails, into lipid regions of the cell membrane1. The labeling buffer provided with the kit (Diluent C) is an iso-osmotic aqueous solution which contains no physiologic salts or buffers, detergents, or organic solvents and is designed to maintain cell viability while maximizing dye solubility and staining efficiency. The labeling efficiency of staining is dependent upon the cell type being labeled and the membranes of the cells2, 3.
NIR Cell Linker Used for Short-term in vitro Studies
CellVue NIR815 dye has been reported to be useful for short-term in vitro cell proliferation studies4, cell tracking in isolated organ preparation applications5, and cell tracking using the Pearl® Small Animal Imaging System or Odyssey® CLx Imager or Odyssey Classic Infrared Imaging System6.
Kit Components
CellVue NIR815 dye stock (1 vial containing 0.1 mL, 1 x 10-3 M in ethanol)
Diluent C (1 vial containing 10 mL, sufficient for 5 labeling reactions)
Excitation and Emission Properties of CellVue NIR815 Dye
The excitation and emission properties of CellVue NIR815 dye are compatible with a range of commercially available near-infrared fluorescent plate readers, flow cytometers, in vitro and in vivo fluorescent imaging systems, and confocal microscopes.
CellVue NIR815 Excitation and Emission Spectra (ex max = 786 nm;em max = 814nm).
Example Data
Imaging of CellVue NIR815 in the capillary region of the lungs of a nude mouse. Image captured with Pearl immediately following intravenous injection into tail vein. Green: CellVue NIR815 cell stain. Red: autofluorescence in animal’s gut detected at 680 nm (caused by chlorophyll in the animal’s diet). Both fluorescent signals are overlaid on a white light image of the mouse.
References
Horan, P. K., and Slezak, S. E., Nature, 340, 167-168 (1989).
Horan, P. K., et al., Methods Cell Biol., 33, 469-490 (1990).
Poon, R.Y., et al. In Living Color: Flow Cytometry and Cell Sorting Protocols, Diamond, R. A., and DeMaggio, S. (Eds.). p. 302-352 (Springer-Verlag, New York, 2000).
Stewart, C. C., Woodring, M. L., Podniesinski, E. and Gray, B. D. Flow Cytometer in the Infrared: Inexpensive Modifications to a Commercial Instrument. Cytometry, Part A, 67A, #2, 104-111 (2005).
Al-Mehdi, A-B, et al. Increased Depth of Cellular Imaging in the Intact Lung Using Far Red and Near Infrared Fluorescent Probes. Int. J. Biomed. Imag., Vol 2006, Article ID 37470, p 1-7.
Thomas, D. L., et al., Experimental Manipulations of Afferent Immune Responses Influence Efferent Immune Responses to Brain Tumors. Cancer Immunol. Immunother.Sep, 57(9): 1323-33 (2008).
CellVue is a registered trademark of PTI Research, Inc, used under license. CellVue products are sold under license from PTI Research, Inc.. US Patent # 8,029,767B2.
When performing quantitative Western blots, internal loading controls and normalization are essential for reliable, precise comparison of protein expression. LI-COR offers several primary antibodies which can be used for two-color normalization when performing multiplex Western blots or for cell-based assay normalization, such as for In-Cell Western™ Assays.
For Western blots, lysate preparation, sample loading, and membrane transfer introduce unavoidable variation. After validation, housekeeping proteins (HKP) can be used for normalization of protein levels.
Normalize and correct for uneven loading, using the intensity of the internal loading control
Visually compare protein levels with confidence, even if you don’t quantify bands
Ensure that visually-observed changes in protein levels represent actual change, not artifacts
Read more about using a housekeeping protein as an internal loading control.
When using an HKP as your normalization strategy, it’s important to validate your HKP for each experiment to ensure its expression is stable. Many factors can influence expression including tissue, treatment, and cell density.
“‘House-keeping’ proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression.”
The Housekeeping Protein Validation Protocol can help you validate that your HKP expression is not changing with treatment. The Housekeeping Protein Normalization Protocol will guide you through the right experimental steps and calculations for normalization using an HKP. Validating your HKP is the only way to know that expression is not affected by the conditions of your experiment
For In-Cell Western Assays, a second protein target (such as actin, tubulin, COX IV, or GAPDH) can be used for normalization. Abundance of the normalization target must be unaffected by the cell treatments used.
Learn more about ICW assay normalization methods.
Comparison of Housekeeping Loading Controls
The table below has a few examples of some common housekeeping proteins, their molecular weights, and a few considerations. This list is not complete, and any housekeeping protein you choose should be fully validated for your system under study.
Western Blot Normalization and Publication Requirements
Sign up to review the science, best practices and mechanics of normalization strategies. Housekeeping proteins can be used as a strategy for Western blot normalization with the right experimental design. Find out what publishers need from you when using the housekeeping protein normalization strategy.
Lambda U® online learning portal is an online, on-demand resources can help ensure that you are getting the very best Western blot data.
Cazzalini O, Sommatis S, Tillhon M, Dutto I, Bachi A, Rapp A, Nardo T, Scovassi AI, Necchi D, Cardoso MC, Stivala LA, Prosperi E. (2014) CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis. Nucleic Acids Res. 42(13): 8433-48.
Lanoix D, St-Pierre J, Lacasse A-A, Viau M, Lafond J, Vaillancourt C. (2012) Stability of reference proteins in human placenta: General protein stains are the benchmark. Placenta. 33: 151–156.
Pérez-Pérez R, López JA, García-Santos E, Camafeita E, Gómez-Serrano M, Ortega-Delgado FJ, Ricart W, Fernández-Real JM, Peral B. (2012) Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity. PLoS ONE 7(1): e30326.
Barber RD, Harmer DW, Coleman RA, Clark BJ. (2005) GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics. 21(3): 389-95.
Rocha-Martins M, Njaine B, Silveira MS. (2012) Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development. PLoS ONE 7(8): e43028.
Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, Wishart TM. (2013) Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting. PLoS ONE 8(8): e72457.
Yu HR, Kuo HC, Huang HC, Huang LT, Tain YL, Chen CC, Liang CD, Sheen JM, Lin IC, Wu CC, Ou CY, Yang KD. (2011) Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in western blot analysis of leukocyte subpopulations from children. Anal Biochem. 413(1): 24–9.
Western Blot Normalization and Publication Requirements
Sign up to review the science, best practices and mechanics of normalization strategies. Housekeeping proteins can be used as a strategy for Western blot normalization with the right experimental design. Find out what publishers need from you when using the housekeeping protein normalization strategy.
Lambda U® online learning portal is an online, on-demand resources can help ensure that you are getting the very best Western blot data.
Chameleon® Vue Pre-stained Protein Ladder,Chameleon® Vue 预染蛋白阶梯
Chameleon® NIR Prestained Protein Ladders,Chameleon® NIR 预染蛋白阶梯
Odyssey® One-Color Protein Molecular Weight Marker,Odyssey® 单色蛋白质分子量标记
WesternSure® Pre-stained Protein Ladder
What is a Protein Marker or Protein Ladder?
A protein marker (also called a protein molecular weight marker, a protein MW marker, or a protein ladder) is used to estimate the size of proteins resolved by gel electrophoresis.
All markers are optimized for use with LI-COR imaging systems but can be used with other imagers.
LI-COR protein ladders and markers are visible on the gel during the run, so you can monitor protein migration.
Markers are used to monitor transfer efficiency from gel to blotting membrane.
* Chameleon Vue can be detected with chemiluminescence when used with the WesternSure Pen, P/N 926-91000
Western Blot Gel Preparation
Sign up to review the science, best practices and mechanics of gel preparation for free, including the use of molecular weight markers at Lambda U® online learning portal.
These online, on-demand resources can help ensure that you are getting the very best Western blot data.
Sign Up
Western Blot Gel Preparation
Sign up to review the science, best practices and mechanics of gel preparation for free, including the use of molecular weight markers at Lambda U® online learning portal.
These online, on-demand resources can help ensure that you are getting the very best Western blot data.
4X Protein Sample Loading Buffer and PVDF Membrane Kit,4X 蛋白质上样缓冲液和 PVDF 膜试剂盒
Protein Sample Loading Buffer for Western Blots
4X Protein Sample Loading Buffer is optimized for use as a loading buffer for protein gel electrophoresis. This orange loading buffer is recommended for use with Odyssey® Imaging Systems as it does not fluoresce in the 700nm channel the way blue loading buffers do. The buffer is optimized for use with SDS-PAGE and Tris-Glycine-SDS running buffer.
Protein Sample Loading Buffer and PVDF Kit for Western Blots
The 4X Protein Sample Loading Buffer and PVDF Membrane Kit comes with 1 roll of Immobilon®-FL PVDF Membrane and 1 bottle of 4X Protein Sample Loading Buffer, 15 mL.
Revert™ 700 Total Protein Stain for Western Blot Normalization,用于蛋白质印迹标准化的 Revert™ 700 总蛋白染色剂
Revert™ 700 Total Protein Stain Kits for Western Blot Normalization,用于蛋白质印迹标准化的 Revert™ 700 总蛋白染色试剂盒
Revert™ 700 Total Protein Stain and Wash Solution Kit,Revert™ 700 总蛋白染色和洗涤液试剂盒
Revert™ 520 Total Protein Stain for Western Blot Normalization,用于蛋白质印迹标准化的 Revert™ 520 总蛋白染色剂
Revert™ 520 Total Protein Stain Kits for Western Blot Normalization,用于蛋白质印迹标准化的 Revert™ 520 总蛋白染色试剂盒
Revert™ 520 Total Protein Stain and Wash Solution Kit,Revert™ 520 总蛋白染色和洗涤液试剂盒
Make Western blot normalization more accurate and reliable with Revert Total Protein Stain, a membrane-based (post-transfer) normalization strategy that stains all protein in your sample. No special reagents, equipment, or gels are required with Revert Total Protein Stains, so they are compatible with your Western blot protocol.
使用 Revert Total Protein Stain 使蛋白质印迹标准化更加准确和可靠，这是一种基于膜的（转移后）标准化策略，可对样品中的所有蛋白质进行染色。 Revert Total Protein Stains 不需要特殊的试剂、设备或凝胶，因此它们与您的蛋白质印迹实验方案兼容。
Use the Gold Standard for Western Blot Normalization
Total protein staining is considered the gold standard for Western blot normalization. Revert Total Protein Stains provide linear signal over a broad range of sample concentrations and are compatible with subsequent Western blot immunodetection methods.
总蛋白染色被认为是蛋白质印迹标准化的金标准。 Revert Total Protein Stains 在广泛的样品浓度范围内提供线性信号，并与随后的蛋白质印迹免疫检测方法兼容。
Quick and Compatible. Stain total protein in less than ten minutes on either PVDF or nitrocellulose membranes.
Accurate Normalization. With a wide linear range of 1 – 60 μg, it’s easy to detect Revert stains and your targets in the same linear range for accurate normalization.
Reliable Analysis. Unlike housekeeping proteins, biological variation won’t affect total protein normalization with Revert Total Protein Staina.
IRDye® 650 Protein Labeling Kits,IRDye® 650 蛋白质标记试剂盒
VRDye™ 549 Protein Labeling Kits,VRDye™ 549 蛋白质标记试剂盒
VRDye™ 490 Protein Labeling Kits,VRDye™ 490 蛋白质标记试剂盒
Visible fluorescence protein labeling kits can be used to label antibodies and other proteins for applications such as flow cytometry, microscopy, immunohistochemistry, and other applications where fluorophore-conjugated antibodies are required.
The visible dyes included in these kits are activated with N-hydroxysuccinimide (NHS) esters, which is the most commonly used reactive group for labeling proteins. NHS esters react with primary amines, forming stable, covalent bonds.
VRDye™ labeling kits are based on a simple conjugation protocol that uses fast, spin column cleanup with resulting purity equivalent to purification by dialysis. Raise the pH of the preservative-free protein solution with the provided buffer, combine it with the appropriate amount of water-soluble amine-reactive dye, and separate the labeled conjugate from the free dye using the included spin columns. Labeling and purification are complete in approximately 2 hours.
Use Visible Fluorescent Dye Conjugates for Microscopy and Flow Cytometry
LI-COR’s visible fluorescent dye products include labeled secondary antibodies and labeling kits.
VRDye™ 549, VRDye 490, and IRDye® 650 secondary antibodies are labeled with visible fluorescent dyes and are optimized for use in Western blotting, microscopy, immunohistochemistry, and flow cytometry applications when imaged on instruments with suitable excitation and emission filters.
These secondary antibodies, like LI-COR IRDye infrared fluorescent secondary antibodies, are highly cross-adsorbed.
The visible fluorescent protein labeling kits are ideal if you need to label your custom monoclonal antibodies for flow cytometry.
Reagents > Chameleon® Vue Pre-stained Protein Ladder
The Chameleon Vue Pre-stained Protein Ladder offers multi-colored, pre-stained proteins for molecular weight estimation. This pre-stained protein ladder:
Allows easy visualization of gel migration and protein size.
Provides a vibrant color scheme that allows you to quickly orient your gel or membrane.
Can be used for chemiluminescent detection when paired with the WesternSure® Pen.
Is optimized for use with Bis-Tris and Tris-Glycine gels.
Not sure which protein marker to choose? Visit How to Choose the Right Protein Ladder.
Representative visible image (left) of the Chameleon Vue Pre-stained Protein Ladder resolved on a 4-12% Bis-Tris Gel and transferred to nitrocellulose via wet tank transfer. The chemiluminescent image (right) was generated using the WesternSure Pen (P/N 926-91000). Pre-stained bands were annotated with the Pen followed by exposure to WesternSure ECL Substrate. The blot was scanned on the C-DiGit® Blot Scanner for 6 minutes.Chameleon Vue 预染色蛋白阶梯的代表性可见图像（左）在 4-12% Bis-Tris 凝胶上分离，并通过湿罐转移转移到硝酸纤维素上。 使用 WesternSure Pen (P/N 926-91000) 生成化学发光图像（右）。 使用 Pen 对预染色条带进行注释，然后暴露于 WesternSure ECL 底物。 在 C-DiGit® Blot Scanner 上扫描印迹 6 分钟。
LI-COR Stripping Buffers Help You Conserve Precious Samples and Time
Stripping and reprobing a Western blot is a common laboratory method. Many labs choose to make their own stripping buffer. With pre-made LI-COR Western blot stripping buffers, you can:
Conserve precious samples by re-using the same blot up to three times to detect different targets or optimize antibody concentrations
Save time and money
Effectively remove antibodies, while retaining immobilized proteins
Strip blots at room temperature in 20 minutes or less without unpleasant odor
Perform qualitative analysis after stripping
NewBlot™ Stripping Buffers are optimized for stripping and reprobing near-infrared (NIR) fluorescent Western blots, including those detected with IRDye® secondary antibodies.
WesternSure® ECL Stripping Buffer is optimized for stripping and reprobing chemiluminescent Western blots.
Use these charcoal canisters with the Pearl® Small Animal Imaging System for in vivo imaging experiments. The canister removes isofluorane gas from the exhaust air stream.
将这些炭罐与 Pearl® Small Animal Imaging System 一起用于体内成像实验。 该罐从废气流中去除异氟烷气体。
Western Blotting Kits with PVDF Membranes,带有 PVDF 膜的蛋白质印迹试剂盒
Western Blotting Kits with Nitrocellulose Membranes,带有硝酸纤维素膜的蛋白质印迹试剂盒
Western Blotting Kits can be used for quantitative Western blots or RNAi studies in which a phosphate-buffered saline (PBS) buffering system or a Tris-buffered saline (TBS) is used.
Convenient Kits for Multiplex Western Blots
These sample kits allow you to obtain a smaller quantity of infrared reagents and corresponding membranes to perform quantitative Western blots without committing to a large quantity of each.
Conveniently test LI-COR reagents and membranes for your Western blotting
Use near-infrared fluorescence detection on the Odyssey® Imaging systems
Experiment with multiplex Western blotting for simultaneous two-color detection
Each kit contains one IRDye® 800CW Dye-Labeled Secondary Antibody for the 800nm channel, plus an additional secondary antibody labeled with IRDye 680RD for the 700nm channel. Kits are available with either 10 Immobilon®-FL PVDF Membranes (0.45 μm, 10 cm x 10 cm) or 10 Odyssey Nitrocellulose Membranes (0.22 μm, 7 cm x 8.5 cm). All kits include Intercept™ (TBS) Blocking Buffer or Intercept (PBS) Blocking Buffer.
每个试剂盒包含一个用于 800nm 通道的 IRDye® 800CW 染料标记二抗，以及一个用于 700nm 通道的用 IRDye 680RD 标记的附加二抗。 套件提供 10 个 Immobilon®-FL PVDF 膜（0.45 μm，10 cm x 10 cm）或 10 个 Odyssey 硝酸纤维素膜（0.22 μm，7 cm x 8.5 cm）。 所有试剂盒均包括 Intercept™ (TBS) 封闭缓冲液或 Intercept (PBS) 封闭缓冲液。
Use Western Blotting Kits for Multiplexing
Dose response of EGFR phosphorylation in A431 cells. A431 cells were treated with serial dilutions of EGF (6.3-200 ng/mL). Lysates were resolved by SDS-PAGE and transferred to nitrocellulose. The blot was probed with rabbit anti-phosphoEGFR and mouse anti-ERK2 followed by detection with IRDye 800CW Goat anti-Rabbit IgG (P/N 926-32211) and IRDye 680RD Goat anti-Mouse IgG (P/N 926-68070).
Optimized Reagents for Chemiluminescent Western Blotting
WesternSure chemiluminescent reagents are optimized for use with film, the C-DiGit® Blot Scanner and the Odyssey® Fc Imaging System. LI-COR chemiluminescent reagents offer the best performance available when compared to other competitive products on the market.
WesternSure PREMIUM Chemiluminescent Substrate is a highly sensitive enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots.
WesternSure HRP-conjugated secondary antibodies are compatible with a variety of chemiluminescent substrates and are optimized for use with WesternSure PREMIUM chemiluminescent substrates.
WesternSure ECL Stripping Buffer allows you to strip and reprobe chemiluminescent Western blots and does not require hazardous shipping, unlike many other stripping buffers.
The WesternSure Pen can be used to annotate any visible protein ladder (such as the Chameleon Vue Ladder) prior to chemiluminescent Western blot detection. The WesternSure Pre-stained Chemiluminescent Protein Ladder is visible on the membrane and reacts with chemiluminescent substrate to emit light.
The Compound Injection Clip helps secure a syringe/catheter system for administering compounds to small animals during imaging. It attaches to the Pearl® Imaging Bed (P/N 9300-21) for easy transfer from work area to instrument.
The Clip accommodates syringes with OD 0.22-0.3 inches and enables injection of compounds even when the imager drawer is closed.
Black Western Blot Incubation Boxes,黑色 Western Blot 培养箱
Pink Western Blot Incubation Boxes,粉色 Western Blot 培养箱
Clear Western Blot Incubation Boxes,透明的 Western Blot 培养箱
Western Blot Incubation boxes are ideal for incubating Western blots, including near-infrared fluorescent Western blots, such as those you would image on the Odyssey® imagers and chemiluminescent Western blots, such as those you would image on the C-DiGit® Blot Scanner or Odyssey XF Dual-Mode Imaging System.
Western Blot Incubation box 非常适合孵育蛋白质印迹，包括近红外荧光蛋白质印迹，例如您将在 Odyssey® 成像仪上成像的蛋白质印迹和化学发光蛋白质印迹，例如您将在 C-DiGit® Blot Scanner 上成像的蛋白质印迹或 Odyssey XF 双模成像系统。
Blot Box Specifications
Four sizes are available in single, five- and ten-pack quantities.
Reagents > COX IV Rabbit Primary Antibody for Normalization
Cytochrome c oxidase is localized to the inner mitochondrial membrane. The COX IV antibody can be used as a mitochondrial loading control and an internal loading control (ILC) and is particularly effective when normalizing low-expressing target proteins.
The expression of COX IV, or any housekeeping protein (HKP), should be validated to ensure that its expression does not change under experimental conditions.
Once validated, COX IV primary antibodies can be used for the detection of COX IV when performing two-color detection.
Detect COX IV Rabbit Monoclonal Primary Antibody with IRDye® Goat anti-Rabbit or IRDye Donkey anti-Rabbit secondary antibodies.
Other options for housekeeping protein normalization
细胞色素 c 氧化酶定位于线粒体内膜。 COX IV 抗体可用作线粒体上样对照和内部上样对照 (ILC)，在标准化低表达靶蛋白时特别有效。
应验证 COX IV 或任何管家蛋白 (HKP) 的表达，以确保其表达在实验条件下不会改变。
一旦通过验证，COX IV 一抗可用于在进行双色检测时检测 COX IV。
使用 IRDye® 山羊抗兔或 IRDye 驴抗兔二抗检测 COX IV 兔单克隆一抗。
管家蛋白质标准化的其他选择
Reactivity and Specificity
COX IV antibody is supplied in 10 mM HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and <0.02% sodium azide.
Do not aliquot the antibody.
COX IV 抗体以 10 mM HEPES (pH 7.5)、150 mM NaCl、100 µg/mL BSA、50% 甘油和 <0.02% 叠氮化钠的形式提供。
不要等分抗体。
Properties
COX IV Rabbit Monoclonal Antibody (P/N 926-42214)
Species Cross-Reactivity
Human, rabbit, monkey, zebrafish, bovine, pig
Target Molecular Weight
17 kDa
Isotype
Rabbit IgG
Specificity/Sensitivity
Detects endogenous levels of total COX IV protein.
Immunogen
A synthetic peptide that corresponds to the residues surrounding Lys29 of human COX IV
Tested Applications
Western blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunofluorescence (IF), Flow Cytometry (F)
This kit contains a replacement desiccant cartridge for the Pearl Imager and the Odyssey® XF Imager.
The Pearl imager and Odyssey XF instrument each have a cooled CCD camera that uses desiccant to prevent water condensation on the internal cooled surfaces.
When the internal relative humidity reaches 30%, the software will issue a warning that the desiccant will soon need to be replaced. When the relative humidity reaches 60%, the cooler will be disabled to prevent condensation.
该套件包含用于 Pearl Imager 和 Odyssey® XF Imager 的替换干燥剂盒。
Pearl 成像仪和 Odyssey XF 仪器各有一个冷却的 CCD 相机，该相机使用干燥剂来防止内部冷却表面上的水凝结。
Reagents > Diluent C for Use with CellVue® Fluorescent Cell Labeling Kits
Diluent C is the labeling buffer required for the dilution of the CellVue NIR815 and CellVue Burgundy dyes. It is an iso-osmotic aqueous solution which contains no physiologic salts or buffers, detergents, or organic solvents and is designed to maintain cell viability while maximizing dye solubility and staining efficiency.
CellVue is a registered trademark of PTI Research, Inc., used under license. Diluent C is sold under license from PTI Research, Inc. US Patents # 8,029,767B2; 7,462,347.
稀释剂 C 是稀释 CellVue NIR815 和 CellVue Burgundy 染料所需的标记缓冲液。 它是一种等渗水溶液，不含生理盐或缓冲液、去污剂或有机溶剂，旨在保持细胞活力，同时最大限度地提高染料溶解度和染色效率。
CellVue 是 PTI Research, Inc. 的注册商标，经许可使用。 稀释剂 C 在 PTI Research, Inc. 美国专利 # 8,029,767B2 的许可下出售； 7,462,347。
Dye contamination found on the surface of animals or within the field of view of the imaging bed will interfere with detection of targets within an animal. External dye contamination can arise from urine residue on the animal’s skin or the imaging bed surface.
Water and alcohol are not very effective for dye residue removal, and depilatory creams can irritate the skin of the animal. This decontamination kit effectively and safely removes dye contamination from the skin of the animal or surface of the Pearl® imaging bed (P/N 9300-21).
GAM CellTag 700 Stain ICW Kits provide detection reagents for cell-based In-Cell Western™ Assays. This cost-effective normalization method makes quantification of the target protein more precise. CellTag 700 Stain accumulates in the nucleus and cytoplasm of permeabilized cells allowing for accurate normalization to cell number.
This kit includes reagents to perform an In-Cell Western assay and CellTag 700 Stain to normalize well-to-well variations in cell number. CellTag 700 accumulates in the nucleus and cytoplasm of permeabilized cells allowing for accurate normalization to cell number.
GAM/GAR CellTag 520 Stain ICW Kits provide detection reagents for cell-based In-Cell Western™ Assays. This cost-effective normalization method makes quantification of the target protein more precise. CellTag 520 Stain accumulates in the nucleus and cytoplasm of permeabilized cells allowing for accurate normalization to cell number.
This kit includes reagents to perform an In-Cell Western Assay and CellTag 520 Stain to normalize well-to-well variations in cell number.
Reagents > GAPDH Rabbit Monoclonal Antibody for Normalization
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a constitutively expressed housekeeping protein (HKP). The GAPDH primary antibody can be used as an internal loading control for normalization.
The expression of GAPDH, or any HKP, should be validated to ensure that its expression does not change under experimental conditions.
Once validated, GAPDH primary antibodies can be used for the detection of GAPDH when performing multiplex Western blot detection.
Detect GAPDH Rabbit Monoclonal Antibody with IRDye® Goat anti-Rabbit or IRDye Donkey anti-Rabbit secondary antibodies.
Other options for housekeeping protein normalization
Reactivity and Specificity
GAPDH antibody is supplied in 10 mM HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and <0.02% sodium azide.
Do not aliquot the antibody.
Properties
GAPDH Rabbit Monoclonal Antibody (P/N 926-42216)
Species Cross-Reactivity
Human, mouse, rat, monkey
Target Molecular Weight
37 kDa
Isotype
Rabbit IgG
Specificity/Sensitivity
Detects endogenous levels of total GAPDH protein. May cross-react with pig
Immunogen
A synthetic peptide that corresponds to residues near the carboxy terminus of human GAPDH
Tested Applications
Western blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)
RRID
P/N 926-42216: RRID AB_2814901
GAPDH Rabbit Monoclonal Antibody in HeLa, NIH/3T3, and COS-7 Lysates
GAPDH Rabbit Monoclonal Antibody detected in HeLa, NIH/3T3, and COS-7 lysates. Lysates were diluted from 2.5 μg to 156 ng. Lysates were separated on 4-12% Bis-Tris gels, electrophoresed at 200V for 45 minutes in MES Running Buffer, and transferred to nitrocellulose membranes in Tris-Glycine buffer at 100V for 65 minutes. Blots were blocked with Intercept® (PBS) Protein-Free Blocking Buffer (P/N 927-90001), probed with GAPDH Rabbit Monoclonal Antibody, and detected on an Odyssey® CLx Imager.
GAR CellTag 700 Stain ICW Kits provide detection reagents for cell-based In-Cell Western™ Assays. This cost-effective normalization method makes quantification of the target protein more precise. CellTag 700 Stain accumulates in the nucleus and cytoplasm of permeabilized cells allowing for accurate normalization to cell number.
This kit includes reagents to perform an In-Cell Western assay and CellTag 700 Stain to normalize well-to-well variations in cell number. CellTag 700 accumulates in the nucleus and cytoplasm of permeabilized cells allowing for accurate normalization to cell number.
GAR/GAM CellTag 520 Stain ICW Kits provide detection reagents for cell-based In-Cell Western™ Assays. This cost-effective normalization method makes quantification of the target protein more precise. CellTag 520 Stain accumulates in the nucleus and cytoplasm of permeabilized cells allowing for accurate normalization to cell number.
This kit includes reagents to perform an In-Cell Western Assay and CellTag 520 Stain to normalize well-to-well variations in cell number.
This filter assembly ensures that the anesthesia flow HEPA-filtered as it flows into the Pearl® Clean Box (P/N 9300-22) or induction chamber. This is for the safety of animals during in vivo imaging experiments on the Pearl Imaging System.
Reagents > Histone H3 Mouse Monoclonal Antibody for Normalization
The Histone H3 primary antibody can be used as an internal loading control for normalization and is particularly effective when detecting target proteins in nuclear extracts.
The expression of Histone H3, or any housekeeping protein (HKP), should be validated to ensure that its expression does not change under experimental conditions.
Once validated, Histone H3 primary antibodies can be used for the detection of Histone H3 when performing multiplex Western blot detection.
Detect Histone H3 Mouse Monoclonal Antibody with IRDye® Goat anti-Mouse or IRDye Donkey anti-Mouse secondary antibodies.
组蛋白 H3 一抗可用作标准化的内部上样对照，在检测核提取物中的靶蛋白时特别有效。
应验证组蛋白 H3 或任何管家蛋白 (HKP) 的表达，以确保其表达在实验条件下不会改变。
经验证后，在进行多重蛋白质印迹检测时，组蛋白 H3 一抗可用于检测组蛋白 H3。
使用 IRDye® 山羊抗小鼠或 IRDye 驴抗小鼠二抗检测组蛋白 H3 小鼠单克隆抗体。
Other options for housekeeping protein normalization
Reactivity and Specificity
Histone H3 antibody is supplied in 10 mM HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and <0.02% sodium azide.
组蛋白 H3 抗体以 10 mM HEPES (pH 7.5)、150 mM NaCl、100 µg/mL BSA、50% 甘油和 <0.02% 叠氮化钠的形式提供。
Detection of Histone H3 Mouse Monoclonal Antibody in HeLa, NIH/3T3, and COS-7 Lysates
Histone H3 Mouse Monoclonal Antibody detected in HeLa, NIH/3T3, and COS-7 Lysates. Lysates were diluted from 2.5 μg to 156 ng. Lysates were separated on 4-12% Bis-Tris gels, electrophoresed at 200V for 45 minutes in MES Running Buffer, and transferred to nitrocellulose membranes in Tris-Glycine buffer at 100V for 65 minutes. Blots were blocked with Intercept® (PBS) Protein-Free Blocking Buffer (P/N 927-90001), probed with Histone H3 Mouse Monoclonal Antibody, and detected on an Odyssey® CLx Imager.在 HeLa、NIH/3T3 和 COS-7 裂解物中检测到组蛋白 H3 小鼠单克隆抗体。 裂解物从 2.5 μg 稀释至 156 ng。 在 4-12% Bis-Tris 凝胶上分离裂解物，在 MES Running Buffer 中以 200V 电泳 45 分钟，然后在 100V 的 Tris-Glycine 缓冲液中转移至硝酸纤维素膜 65 分钟。 印迹用 Intercept® (PBS) 无蛋白封闭缓冲液（P/N 927-90001）封闭，用组蛋白 H3 小鼠单克隆抗体探测，并在 Odyssey® CLx 成像仪上检测。
Reagents > Histone H3 Rabbit Monoclonal Antibody for Normalization
The Histone H3 primary antibody can be used as an internal loading control for normalization and is particularly effective when detecting target proteins in nuclear extracts.
The expression of Histone H3, or any housekeeping protein (HKP), should be validated to ensure that its expression does not change under experimental conditions.
Once validated, Histone H3 primary antibodies can be used for the detection of Histone H3 when performing multiplex Western blot detection.
Detect Histone H3 Rabbit Monoclonal Antibody with IRDye® Goat anti-Rabbit or IRDye Donkey anti-Rabbit secondary antibodies.
组蛋白 H3 一抗可用作标准化的内部上样对照，在检测核提取物中的靶蛋白时特别有效。
应验证组蛋白 H3 或任何管家蛋白 (HKP) 的表达，以确保其表达在实验条件下不会改变。
经验证后，在进行多重蛋白质印迹检测时，组蛋白 H3 一抗可用于检测组蛋白 H3。
使用 IRDye® 山羊抗兔或 IRDye 驴抗兔二抗检测组蛋白 H3 兔单克隆抗体。
Other options for housekeeping protein normalization
Reactivity and Specificity
Histone H3 antibody is supplied in 10 mM HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and <0.02% sodium azide.
Detects endogenous levels of total Histone H3 protein (including isoforms H3.1, H3.2, and H3.3) and Histone H3 variant CENP-A. Does not cross-react with other core histones. May cross-react with bovine, chicken, D. melanogaster, hamster, xenopus, and zebrafish.
Immunogen
A synthetic peptide that corresponds to the carboxy terminus of the human histone H3 protein
Tested Applications
Western blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Flow Cytometry (F)
蛋白质印迹 (WB)、免疫组织化学 (IHC)、免疫荧光 (IF)、流式细胞术 (F)
RRID
P/N 926-42219: RRID AB_2814902
Detection of Histone H3 Rabbit Monoclonal Antibody in HeLa, NIH/3T3, and Cos7 Lysates
Histone H3 Rabbit Monoclonal Antibody detected in HeLa, NIH/3T3, and COS-7 Lysates. Lysates were diluted from 2.5 μg to 156 ng. Lysates were separated on 4-12% Bis-Tris gels, electrophoresed at 200V for 45 minutes in MES Running Buffer, and transferred to nitrocellulose membranes in Tris-Glycine buffer at 100V for 65 minutes. Blots were blocked with Intercept® (PBS) Protein-Free Blocking Buffer (P/N 927-90001), probed with Histone H3 Rabbit Monoclonal Antibody, and detected on an Odyssey® CLx Imager.在 HeLa、NIH/3T3 和 COS-7 裂解物中检测到组蛋白 H3 兔单克隆抗体。 裂解物从 2.5 μg 稀释至 156 ng。 在 4-12% Bis-Tris 凝胶上分离裂解物，在 MES Running Buffer 中以 200V 电泳 45 分钟，然后在 100V 的 Tris-Glycine 缓冲液中转移至硝酸纤维素膜 65 分钟。 印迹用 Intercept® (PBS) 无蛋白封闭缓冲液（P/N 927-90001）封闭，用组蛋白 H3 兔单克隆抗体探测，并在 Odyssey® CLx 成像仪上检测。
This is an optional analysis key available for purchase separately with the following software:
Image Studio™ Software for the C-DiGit® Blot Scanner
Image Studio Lite Software
With this key, you can analyze in vitro assays in a microplate format. For 96-well or 384-well microplates, circles are automatically placed over your wells. You can also perform ratiometric calculations.
Note: The C-DiGit Blot Scanner cannot image microplates, but the software can import images acquired with other LI-COR instruments for analysis.
This is an optional key available for purchase for use with Image Studio™ Software for the Odyssey® CLx Imager or Odyssey Classic Imager.
With this key, you can perform advanced analyses for In-Cell Western assays, such as cell count normalization, percent response, and Z′-Factor analysis.
Try Intercept (PBS) Blocking Buffer and Intercept T20 (PBS) Antibody Diluent for improved results in your quantitative Western blots and other immunoassays. Intercept Blocking Buffers provide excellent blocking performance with low background and low variability.
Intercept T20 Antibody Diluents improve the specificity of the primary and secondary antibodies, reducing off-target effects. They are preformulated with Tween® 20. There’s no need to mix the diluent yourself, which saves you time and reduces potential variation.
Try Intercept (TBS) Blocking Buffer and Intercept T20 (TBS) Antibody Diluent for improved results in your quantitative Western blots and other immunoassays. Intercept Blocking Buffers provide excellent blocking performance with low background and low variability.
Intercept T20 Antibody Diluents improve the specificity of the primary and secondary antibodies, reducing off-target effects. They are preformulated with Tween® 20. There’s no need to mix the diluent yourself, which saves you time and reduces potential variation.
Intercept T20 (PBS) Antibody Diluent improves the specificity of the primary and secondary antibodies, reducing off-target effects.The diluent contains Intercept (PBS) Blocking Buffer preformulated with Tween® 20 for use in a phosphate-buffered saline (PBS) system.
Since there’s no need to mix the diluent yourself, it saves you time and reduces potential variation.
Formulation
Intercept T20 (PBS) Antibody Diluent contains a 0.05% concentration of Tween 20. It contains no sodium azide and is stored at 4 °C.
Shake well before each use.
Intercept T20 (PBS) Antibody Diluent can be used for many immunoassays and applications, including:
Isolation of specific antibodies was accomplished by affinity chromatography using antigens immobilized on agarose beads. Based on immunoelectrophoresis and/or ELISA, the antibody reacts with whole molecule guinea pig IgG as well as the light chains common to other guinea pig immunoglobulins. No reactivity was detected against non-immunoglobulin serum proteins. This antibody has been tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, chicken, goat, Syrian hamster, horse, human, mouse, rabbit, rat, and sheep serum proteins. The conjugate has been specifically tested and qualified for Western blot applications.
IRDye 680LT secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Optimum dilutions will vary and should be determined empirically.
Note: When using PVDF membranes for Western blotting applications, SDS (final concentration of 0.01 – 0.02%) and Tween® 20 (final concentration of 0.1 – 0.2%) must be added during the detection incubation step to avoid non-specific background staining.
Detection of guinea pig IgG with IRDye 680LT Donkey anti-Guinea Pig. Serial dilutions of Guinea Pig IgG were spiked into C32 lysate (Santa Cruz P/N sc-2205). Samples were resolved on a 10% Bis-Tris gel and transferred to Odyssey® Nitrocellulose membrane (P/N 926-31092). The membrane was blocked with Odyssey Blocking Buffer (P/N 927-40000) followed by detection with IRDye 680LT Donkey anti-Guinea Pig (P/N 926-68030).
Isolation of specific antibodies was accomplished by affinity chromatography on mouse IgG2b covalently linked to agarose. Based on ELISA and flow cytometry, the antibody reacts with the heavy chain of mouse IgG2b. This antibody was tested by dot blot and/or solid-phase adsorbed to ensure minimal cross-reactivity with mouse IgM, IgG1, IgG2a, IgG3, and IgA, pooled human sera, and purified human paraproteins. The conjugate has been specifically tested and qualified for Western blot applications. Results with your primary antibody may vary and specificity should be confirmed prior to performing two-color detection.
Applications
Recommended for:
Western Blot
Protein Array
Immunohistochemistry
Microscopy
2D Gel Detection
Tissue Section Imaging
Not Recommended for:
Small Animal Imaging
In-Cell Western Assay
On-Cell Western Assay
Formulation
IRDye 680LT secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Optimum dilutions will vary and should be determined empirically.
Note: When using PVDF membranes for Western blotting applications, SDS (final concentration of 0.01 – 0.02%) and Tween® 20 (final concentration of 0.1 – 0.2%) must be added during the detection incubation step to avoid non-specific background staining.
RRID
P/N 926-68052: RRID AB_2783644
Example Data
Western blot detection of p53 in various lysates. Lanes 1-5: Odyssey® One-Color Molecular Weight Marker (P/N 928-40000), Hela, COS7, A431, and C32 cell lysates. The membrane was blocked with Odyssey Blocking Buffer (P/N 927-40000) and probed with mouse anti-p53 (IgG2a) (Santa Cruz P/N sc-126) and Mouse anti-β Actin (IgG2b) (P/N 926-42212) followed by detection with IRDye 800CW Goat anti-Mouse IgG2a (P/N 926-32351) and IRDye 680LT Goat anti-Mouse IgG2b (P/N 926-68052).各种裂解物中 p53 的蛋白质印迹检测。 泳道 1-5：Odyssey® One-Color Molecular Weight Marker (P/N 928-40000)、Hela、COS7、A431 和 C32 细胞裂解物。 用 Odyssey 封闭缓冲液 (P/N 927-40000) 封闭膜，并用小鼠抗 p53 (IgG2a) (Santa Cruz P/N sc-126) 和小鼠抗β肌动蛋白 (IgG2b) (P/N 926) 进行探测 -42212)，然后使用 IRDye 800CW 山羊抗小鼠 IgG2a (P/N 926-32351) 和 IRDye 680LT 山羊抗小鼠 IgG2b (P/N 926-68052) 进行检测。
Isolation of specific antibodies was accomplished by affinity chromatography on mouse IgG1 covalently linked to agarose. Based on ELISA and flow cytometry, the antibody reacts with the heavy chain of mouse IgG1. This antibody has been tested by dot blot and/or solid-phase adsorbed to ensure minimal cross-reactivity with mouse IgM, IgG2a, IgG2b, IgG3, and IgA, pooled human sera, and purified human paraproteins. The conjugate has been specifically tested and qualified for Western blot applications. Results with your primary antibody may vary and specificity should be confirmed prior to performing two-color detection.
IRDye 680LT secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Optimum dilutions will vary and should be determined empirically.
Note: When using PVDF membranes for Western blotting applications, SDS (final concentration of 0.01 – 0.02%) and Tween® 20 (final concentration of 0.1 – 0.2%) must be added during the detection incubation step to avoid non-specific background staining.
Western blot analysis of PTEN expression in mouse PTEN transfected 293T whole cell lysate (lane 2) and non-transfected 293T lysate (lane 3). Both lysates were loaded with 2 µg total protein per lane. Odyssey® One-Color Molecular Weight Marker (P/N 928-40000) was loaded in lane 1. The blot was probed with mouse ant-PTEN (IgG2b) (Santa Cruz P/N sc-133197) and mouse anti-GAPDH (IgG1) (Abcam P/N ab8245) followed by detection with IRDye 800CW Goat anti-Mouse IgG2b (P/N 926-32352) and IRDye 680LT Goat anti-Mouse IgG1 (926-68050)
Isolation of specific antibodies was accomplished by affinity chromatography on mouse IgG2a covalently linked to agarose. Based on ELISA and flow cytometry, this antibody reacts with the heavy chain of mouse IgG2a. This antibody has been tested by dot blot and/or solid-phase adsorbed to ensure minimal cross-reactivity with mouse IgM, IgG1, IgG2b, IgG3, and IgA, pooled human sera, and purified human paraproteins. The conjugate has been specifically tested and qualified for Western blot. Results with your primary antibody may vary and specificity should be confirmed prior to performing two-color detection.
IRDye 680LT secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Optimum dilutions will vary and should be determined empirically.
Note: When using PVDF membranes for Western blotting applications, SDS (final concentration of 0.01 – 0.02%) and Tween® 20 (final concentration of 0.1 – 0.2%) must be added during the detection incubation step to avoid non-specific background staining.
IRDye 680LT Maleimide will label molecules with free sulfhydryl (–SH) groups, such as cysteine residues in proteins. Conjugation can be performed at physiological pH.
Which 700 Channel Dye Should I Use?
Note: IRDye 680LT dye products should not be used for small animal in vivo imaging or In‑Cell Western™ Assays. The higher level of fluorescent intensity creates high background making it unfavorable for use in these applications. We recommend using IRDye 680RD dye products for these applications.
IRDye infrared dyes from LI-COR Biosciences are available for researchers developing applications using near-infrared (NIR) fluorescence.
Standard NHS ester chemistry is used to produce custom probes labeled with LI-COR IRDye infrared dyes. The NHS ester reactive group provides the functionality for labeling primary and secondary amines, such as lysine residues in proteins.
Which 700 Channel Dye Should I Use?
Note: IRDye 680LT dye products should not be used for small animal in vivo imaging or In‑Cell Western™ Assays. The higher level of fluorescent intensity creates high background making it unfavorable for use in these applications. We recommend using IRDye 680RD dye products for these applications.
IRDye 680LT Streptavidin is supplied as a liquid in buffer containing 10 mM phosphate, 183 mM NaCl, 2.7 nM KCl, pH 7.4 with sodium azide 0.005% (w/v) as a preservative.
To use, centrifuge briefly before use to eliminate aggregates that may have formed in solution. This will reduce non-specific background staining. A final concentration of 0.2 to 1.0 µg/ml (1:1,000 to 1:5,000) is usually satisfactory for most applications; however, appropriate dilution may need to be determined empirically.
For membrane-based applications and In-Gel Westerns, it is recommended to add SDS (0.02% to 0.1% final concentration), in addition to Tween® 20 (0.1 to 0.2% final concentration) during the detection incubation step to reduce non-specific background staining.
IRDye 680LT 链霉亲和素以液体形式提供，缓冲液中含有 10 mM 磷酸盐、183 mM NaCl、2.7 nM KCl，pH 7.4，叠氮化钠 0.005% (w/v) 作为防腐剂。
Assays that use IRDye 680RD conjugates (such as small animal imaging and cell binding assays) may require a “dye-only” control for potential effects or retention of the dye.
Carboxylate (non-reactive) form of IRDye 680RD is an ideal control.
Note: The carboxylate dye has no reactive group and cannot be used for labeling.
Chicken IgY, whole molecule. IgY is the original designation for the IgG-like protein found in both serum and egg yolk.
Purity and Specificity
The antibody was isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. Based on immunoelectrophoresis and/or ELISA, this antibody reacts with whole molecule chicken IgY, and with the light chains of other chicken immunoglobulins. No reactivity was detected against non-immunoglobulin serum proteins. This antibody was tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, goat, guinea pig, Syrian hamster, horse, human, mouse, rabbit, rat, and sheep serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Isolation of specific antibodies was accomplished by affinity chromatography using pooled IgG covalently linked to agarose. Based on ELISA, this antibody reacts with the heavy and light chains of goat IgG and with sheep IgG. This antibody was tested by dot blot and/or solid-phase adsorbed to ensure minimal cross-reactivity with human, mouse, rabbit, rat, chicken. guinea pig, hamster, swine, and horse serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
Applications
Highly recommended for:
Western Blot
In-Cell Western Assay
On-Cell Western Assay
Protein Array
Immunohistochemistry
Small Animal Imaging
Microscopy
2D Gel Detection
Tissue Section Imaging
Formulation
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Optimum dilutions will vary and should be determined empirically.
Note: Do not use this secondary antibody in combination with any secondary antibody whose host is goat (for example, IRDye 800CW Goat anti-Rabbit) for two-color Western blot detection.
Isolation of specific antibodies was accomplished by immunoaffinity chromatography using antigens immobilized on agarose beads. Based on immunoelectrophoresis and/or ELISA, this antibody reacts with whole molecule guinea pig IgG, and with the light chains common to other guinea pig immunoglobulins. This antibody was tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, chicken, goat, Syrian hamster, horse, human, mouse, rabbit, rat, and sheep serum proteins. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
The antibody was isolated by affinity chromatography using antigens coupled to agarose beads. Based on immunoelectrophoresis, this antibody reacts with the heavy chains of mouse IgG, and with the light chains common to most mouse immunoglobulins. No reactivity was detected against non-immunoglobulin serum proteins. This antibody was tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, chicken, goat, guinea pig, Syrian hamster, horse, human, rabbit, and sheep serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
The antibody was isolated by affinity chromatography using antigens coupled to agarose beads. Based on ELISA, this antibody reacts with the heavy and light chains of rabbit IgG, and with the light chains common to most rabbit immunoglobulins. This antibody was tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, chicken, goat, guinea pig, hamster, horse, human, mouse, rat, and sheep serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Isolation of specific antibodies was accomplished by affinity chromatography using human IgG covalently linked to agarose. Based on ELISA, this antibody reacts with the heavy and light chains of human IgG. This antibody was tested by dot blot and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, horse, and mouse serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Isolation of specific antibodies was accomplished by affinity chromatography using pooled mouse IgG covalently linked to agarose. Based on ELISA and flow cytometry, this antibody reacts with the heavy and light chains of mouse IgG1, IgG2a, IgG2b, and IgG3, and with the light chains of mouse IgM and IgA. This antibody was tested by dot blot and and/or solid-phase adsorbed for minimal cross-reactivity with human, rabbit, goat, rat, and horse serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Isolation of specific antibodies was accomplished by affinity chromatography using mouse IgM covalently linked to agarose. Based on ELISA and/or flow cytometry, this antibody reacts with the heavy chain of mouse IgM. This antibody was tested by dot blot and/or solid-phase adsorbed to ensure minimal cross-reactivity with mouse IgG1, IgG2a, IgG3, and IgA, pooled human sera and purified human paraproteins. The conjugate has been specifically tested and qualified for Western blot applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
Isolation of specific antibodies was accomplished by affinity chromatography using pooled rabbit IgG covalently linked to agarose. Based on ELISA and flow cytometry, this antibody reacts with the heavy and light chains of rabbit IgG, and with the light chains of rabbit IgM and IgA. This antibody was tested by dot blot and and/or solid-phase adsorbed for minimal cross-reactivity with human, mouse, rat, sheep, and chicken serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
The antibody was purified from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. Based on immunoelectrophoresis and/or ELISA, this antibody reacts with whole molecule rat IgG, and with the light chains of other rat immunoglobulins. No reactivity was detected against non-immunoglobulin serum proteins. This antibody was tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with mouse, bovine, horse, human, and rabbit serum proteins, but may cross-react with immunoglobulins from other species. The conjugate has been specifically tested and qualified for Western blot and In-Cell Western™ assay applications.
IRDye 680RD secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.
Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.
IRDye 680RD Maleimide will label molecules with free sulfhydryl (-SH) groups, such as cysteine residues in proteins. Conjugation can be performed at physiological pH.
IRDye infrared dyes from LI-COR Biosciences are available for researchers developing applications using near-infrared (NIR) fluorescence. These unique dyes are used in a variety of NIR imaging applications including Western blotting, plate-based assays, protein arrays, tissue section imaging, and molecular activity measurements.
Standard NHS ester chemistry is used to produce custom probes labeled with LI-COR IRDye infrared dyes. The NHS ester reactive group provides the functionality for labeling primary and secondary amines, such as lysine residues in proteins.
NHS Ester reacts with a primary aliphatic amine, such as lysine.Dye label is attached through amide bond.
Reagents > IRDye® 680RD Protein Labeling Kits,IRDye® 680RD 蛋白质标记试剂盒
IRDye 680RD Protein Labeling Kits can be used to label antibodies and other proteins for Western blotting, In-Cell Western™ assays, in vivo imaging, and whole organ or tissue section applications.
IRDye 680RD Streptavidin is supplied as a liquid in buffer containing 10 mM phosphate, 183 mM NaCl, 2.7 nM KCl, pH 7.4 with sodium azide 0.005% (w/v) as a preservative.
To use, centrifuge briefly before use to eliminate aggregates that may have formed in solution. This will reduce non-specific background staining. A final concentration of 0.2 to 1.0 µg/ml (1:1,000 to 1:5,000) is usually satisfactory for most applications; however, appropriate dilution may need to be determined empirically.
For membrane-based applications and In-Gel Westerns, it is recommended to add SDS (0.02% to 0.1% final concentration), in addition to Tween® 20 (0.1 to 0.2% final concentration) during the detection incubation step to reduce non-specific background staining.
IRDye 680RD 链霉亲和素以液体形式提供，缓冲液中含有 10 mM 磷酸盐、183 mM NaCl、2.7 nM KCl，pH 7.4，叠氮化钠 0.005% (w/v) 作为防腐剂。
Underlined nucleotides are the binding site. IRDye 700 oligonucleotides are supplied as 25 µL of 50 nM (or 50 fmol/µL) double-stranded DNA.
AP-1 is a transcription factor that regulates gene expression and controls a number of cellular processes including differentiation, proliferation, and apoptosis. For this reason, it is a common oligo used in EMSA to assess protein binding.
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 AP-1 oligonucleotide, the following binding reaction is a good starting point:
Reaction
µL
10X Binding Buffer (100 mM Tris, 500 mM KCI, 10 mM DTT; pH 7.5)
2
Poly (dl•dC) 1 µg/µL in 10 mM Tris, 1 mM EDTA; pH 7.5
After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature.
Since IRDye infrared dyes are somewhat sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put the tubes into a drawer or simply cover the rack containing tubes with aluminum foil). After the incubation period, 10X Orange Loading Dye (P/N 927-10100) is added to the binding reaction for electrophoresis.
将 DNA 添加到蛋白质缓冲液混合物中后，孵育反应物以使蛋白质与 DNA 结合。 典型的孵育条件是室温下 20-30 分钟。
Underlined nucleotides are the binding site. IRDye 700 oligonucleotides are supplied as 25 µL of 50 nM (or 50 fmol/µL) double-stranded DNA.
CREB proteins are cellular transcription factors activated by phosphorylation of various kinases that bind to certain DNA sequences. For this reason, it is a common oligonucleotide used in EMSA to assess protein binding.
CREB 蛋白是由与某些 DNA 序列结合的各种激酶的磷酸化激活的细胞转录因子。 出于这个原因，它是 EMSA 中用于评估蛋白质结合的常见寡核苷酸。
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 CREB oligonucleotide, the following binding reaction is a good starting point:
Reaction
µL
10X Binding Buffer (100 mM Tris, 500 mM KCI, 10 mM DTT; pH 7.5)
2
Poly (dl•dC) 1 µg/µL in 10 mM Tris, 1 mM EDTA; pH 7.5
1
25 mM DTT/2.5% Tween® 20
2
1% NP-40
1
Water
12
IRDye 700 CREB
1
PC12 nuclear extract (positive control) (5 µg/µL) in Dilution Buffer (20 mM Hepes (pH 7.9), 100 mM KCI, 1 mM MgCl2, 20% glycerol, 0.5 mM PMSF and 0.5 mM DTT)
1
Total
20
After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature.
Since IRDye infrared dyes are somewhat sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put the tubes into a drawer or simply cover the rack containing tubes with aluminum foil). After the incubation period, 10X Orange Loading Dye (P/N 927-10100) is added to the binding reaction for electrophoresis.
将 DNA 添加到蛋白质缓冲液混合物中后，孵育反应物以使蛋白质与 DNA 结合。 典型的孵育条件是室温下 20-30 分钟。
Underlined nucleotides are the binding site. IRDye 700 oligonucleotides are supplied as 25 µL of 50 nM (or 50 fmol/µL) double-stranded DNA.
HIF-1 is a transcription factor that regulates celluar responses to hypoxia, which have been heavily implicated in cancer biology and various other pathophysiologies. For this reason, it is a common oligonucleotide used in EMSA assays in basic discovery and therapeutics research.
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 HIF-1 oligonucleotide, the following binding reaction is a good starting point:
Reaction
µL
10X Binding Buffer (100 mM Tris, 500 mM KCI, 10 mM DTT; pH 7.5)
2
Poly (dl•dC) 1 µg/µL in 10 mM Tris, 1 mM EDTA; pH 7.5
1
25 mM DTT/2.5% Tween® 20
2
Water
13
IRDye 700 HIF-1
1
COS-7 (CoCl2 TREATED) nuclear extract – (positive control) (5 µg/µL) in Dilution Buffer (20 mM Hepes (pH 7.9), 100 mM KCI, 1 mM MgCl2, 20% glycerol, 0.5 mM PMSF, and 0.5 mM DTT)
1
Total
20
After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature.
Since IRDye infrared dyes are somewhat sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put the tubes into a drawer or simply cover the rack containing tubes with aluminum foil). After the incubation period, 10X Orange Loading Dye (P/N 927-10100) is added to the binding reaction for electrophoresis.
将 DNA 添加到蛋白质缓冲液混合物中后，孵育反应物以使蛋白质与 DNA 结合。 典型的孵育条件是室温下 20-30 分钟。
Underlined nucleotides are the binding site. IRDye 700 oligonucleotides are supplied as 25 µL of 50 nM (or 50 fmol/µL) double-stranded DNA.
NFκB is a transcription factor that controls transcription of DNA, cytokine production, and cell survival. For this reason, it is a common oligonucleotide used in EMSA to assess the binding of specific proteins.
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 NFĸB oligonucleotide, the following binding reaction is a good starting point:
Reaction
µL
10X Binding Buffer (100 mM Tris, 500 mM KCI, 10 mM DTT; pH 7.5)
2
Poly (dl•dC) 1 µg/µL in 10 mM Tris, 1 mM EDTA; pH 7.5
1
25 mM DTT/2.5% Tween® 20
2
1% NP-40
1
Water
12
IRDye 700 NFĸB
1
Raji nuclear extract (positive control) (5 µg/µL) in Dilution Buffer (20 mM Hepes (pH 7.9), 100 mM KCI, 1 mM MgCl2, 20% glycerol, 0.5 mM PMSF, and 0.5 mM DTT)
1
Total
20
After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature.
Since IRDye infrared dyes are somewhat sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put the tubes into a drawer or simply cover the rack containing tubes with aluminum foil). After the incubation period, 10X Orange Loading Dye (P/N 927-10100) is added to the binding reaction for electrophoresis.
Other IRDye 700 oligonucleotides as well as IRDye 800 oligonucleotides are available through Integrated DNA Technologies, TriLink BioTechnologies, or Metabion International AG.
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