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Pierce S-Nitrosylation Western Blot Kit


A convenient and sensitive tool for detecting S-nitrosylated protein modifications.

Pierce S-Nitrosylation Western Blot Kit

The Thermo Scientific Pierce S-Nitrosylation Western Blot Kit enables sensitive detection of protein S-nitrosocysteine post-translational modifications in a complete, easy-to-use kit.

The Pierce S-Nitrosylation Western Blot Kit contains a cell lysis/reaction buffer, a sulfhydryl-reactive blocking agent, a reducing agent, a labeling agent and a detection monoclonal antibody. Each kit has enough reagents to label 40 samples of 100µg each. Similar to the traditional S-nitrosylation switch assay, unmodified cysteines are first blocked using a sulfhydryl-reactive compound (MMTS). S-nitrosylated cysteines are then selectively reduced with ascorbate in HENS Buffer for specific labeling with iodoTMTzero™ Reagents, which irreversibly bind to the cysteine thiol that was S-nitrosylated. Detection of the TMT™ reagent-modified proteins is facilitated using an anti-TMT antibody.

Highlights:

  • Specific – optimized labeling reactions switch S-nitrosocysteine post translational modifications with iodoTMT Reagent for easy detection by Western blot using anti-TMT antibody
  • Better signal-to-noise – uses a non-biological iodoTMT Reagent for labeling instead of HPDP-biotin, resulting in less background during Western blot detection
  • Workflow-compatible – labeling method is compatible with alternative methods of analysis, including enrichment via anti-TMT resin and multiplex quantitation by mass spectrometry
  • Convenient – all needed reagents included one kit

Includes:

  • Cysteine-blocking reagent, reducing agent, reaction buffer, iodoTMT™ labeling reagent, and anti-TMT antibody

Product Details:

S-nitrosylation is a post-translational modification that regulates numerous processes, including cellular proliferation, apoptosis, smooth muscle relaxation, neurotransmitter release, and differentiation (Ref.1). During S-nitrosylation, nitric oxide radicals react with cysteine thiols to produce an S-NO adduct that alters protein activity.

In 1998, Jaffrey et al. described the biotin switch assay, which used HPDP-biotin to measure in vivo and in vitro S-nitrosylation (Ref.2). In this assay, free sulfhydryls are chemically blocked, whereas nitrosylated cysteines are selectively reduced and biotinylated for Streptavidin-HRP Western blot detection. However, most cells produce biotinylated polypeptides which yield false-positive signals during Western blot analysis. In addition, the biotin labeling chemistry is reversible, preventing S-nitrosylation site identification by mass spectrometry.

The Pierce S-Nitrosylation Western Blot Kit provides all of the necessary reagents for a modified form of the traditional S-nitrosylation switch assay. Labeling is accomplished using the non-biological iodoTMT™ Reagent instead of HPDP-biotin, and this results in less background during Western blot detection. In addition to detection of S-nitrosylated proteins by Western blot, an immobilized anti-TMT resin can be used to selectively enrich S-nitrosylated proteins/peptides labeled with iodoTMT reagents. This workflow allows for S-nitrosylation site mapping and multiplexed quantititation using mass spectrometry.

Reaction scheme for labeling and detection of S-nitrosylation with iodoTMT reagent
Reaction scheme for labeling and detection of S-nitrosylation with Thermo Scientific Pierce S-Nitrosylation Western Blot Kit. Samples are first reacted with MMTS to block free sulfhydryls in S-nitrosylated proteins. The S-nitrosocysteines are then selectively reduced with ascorbate for labeling with the iodoTMTzero reagent. Subsequently, the supplied anti-TMT antibody is used to detect the TMT-labeled proteins in a Western blot.

 

Lower background than biotin-switch assay for S-nitrosylation.
Lower background with iodoTMT reagent compared to biotin labeling for detection of S-nitrosylated proteins in A549 cell lysate. Protein samples were either not treated or S-nitrosylated using S-nitroso-glutathione. Following S-nitrosylation (NO) treatment, all samples were blocked with MMTS before labeling using iodoTMT reagent (left panel) or HPDP-biotin (right panel) in the absence or presence of ascorbate. Sample were separated by SDS-PAGE and blotted to membrane for Western blot detection. The TMT-labeled samples were detected by the anti-TMT antibody and biotin-labeled samples were detected by streptavidin-conjugated HRP. In a perfect assay method, nothing would be detected in the first three lanes of each blot; nonspecific background is much higher in the biotin-based experiment.

 

More Product Data

Cited References:

  1. Jaffrey, S.R., et. al. (2001). Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biology. 3:193-7.
  2. Foster, M. and J.S. Stamler. (2004). New insights into protein S-nitrosylation. J. Biol. Chem. 279: 25891-7.
  3. Foster, M.W., D.T. Hess, and J.S. Stamler. (2009). Protein S-nitrosylation in health and disease: a current perspective. Trends Mol. Med. 15:391-404.

Related Products:

Tandem Mass Tag™ Reagents
Cysteine-reactive TMT Reagents
EZ-Link HPDP-Biotin
MMTS - Methyl methanethiosulfonate


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
90105 S-Nitrosylation Western Blot Kit
Formulation: Multi-component kit
Sufficient For: 40 labeling reactions of 100μg each

Kit Contents:
iodoTMTzero Label Reagent, 2 x 0.2mg
HENS Buffer, 100mL
Methyl Methanethiosulfonate (MMTS), 200mg
Sodium Ascorbate, 20mg
Anti-TMT Antibody, 100μL
40-rxn kit Product Instructions for product #90105 S-Nitrosylation Western Blot Kit MSDS for product #90105 S-Nitrosylation Western Blot Kit Certificate of Analysis for product #90105 S-Nitrosylation Western Blot Kit $308.00

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90106 HENS Buffer
Formulation: 100mM HEPES, pH 7.8; 1mM EDTA, 0.1mM neocuproine, 1% SDS
Sufficient For: Detection of the TMT label in Western blots
100mL Product Instructions for product #90106 HENS Buffer MSDS for product #90106 HENS Buffer Certificate of Analysis for product #90106 HENS Buffer $77.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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