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.
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