Pierce™ S-Nitrosylation Western Blot Kit and HENS Buffer

The Pierce S-Nitrosylation Western Blot Kit provides cell lysis/reaction buffer, sulfhydryl-reactive blocking agent, reducing agent, labeling agent, and anti-TMT antibody to label 40 samples (100 μg each). As with traditional S-nitrosylation switch assays, unmodified cysteines are blocked by a sulfhydryl-reactive compound. S-nitrosylated cysteines are selectively reduced with ascorbate in HENS Buffer for 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. During S-nitrosylation, nitric oxide radicals react with cysteine thiols to produce an S-NO adduct that alters protein activity.

Pierce S-Nitrosylation Western Blot Kit
The Pierce S-Nitrosylation Western Blot Kit provides the necessary reagents to perform a modified version of the traditional S-nitrosylation switch assay, including a cysteine-blocking reagent, reducing agent, reaction buffer, iodoTMT labeling reagent, and anti-TMT antibody. Labeling is accomplished using the non-biological iodoTMT reagent instead of HPDP-biotin, reducing background signal 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 quantitation using mass spectrometry.

Features of Pierce S-Nitrosylation Western Blot Kit include:
• 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

HENS Buffer
HENS Buffer consists of 100 mM HEPES (pH 7.8), 1 mM EDTA, 0.1 mM Neocuproine, and 1% SDS. It is a cell lysis buffer intended for use with the Pierce S-Nitrosylation Western Blot Kit, which enables sensitive detection of protein S-nitrosocysteine post-translational modifications in a complete, easy-to-use kit.