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.
Features of Pierce S-Nitrosylation Western Blot Kit:
—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
—labeling method is compatible with alternative methods of analysis, including enrichment via anti-TMT resin and multiplex quantitation by mass spectrometry
—all needed reagents included one kitIncludes:
• Cysteine-blocking reagent, reducing agent, reaction buffer, iodoTMT™ labeling reagent, and anti-TMT antibody
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 100 µg 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.
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.More Product Data
• Specific labeling, enrichment and quantitation of S-nitrosylated peptides using iodoTMT reagentsRelated ProductsHENS Buffer for Pierce™ S-Nitrosylation Western Blot Kit
For Research Use Only. Not for use in diagnostic procedures.