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.
- 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
- Cysteine-blocking reagent, reducing agent, reaction buffer, iodoTMT™ labeling reagent, and 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.
|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 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.
- Jaffrey, S.R., et. al. (2001). Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biology. 3:193-7.
- Foster, M. and J.S. Stamler. (2004). New insights into protein S-nitrosylation. J. Biol. Chem. 279: 25891-7.
- 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.
Tandem Mass Tag™ Reagents
Cysteine-reactive TMT Reagents
MMTS - Methyl methanethiosulfonate