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Nitric oxide (NO) is produced by three distinct isoforms of nitric oxide synthase and functions as a key signaling molecule in physiology and pathophysiology. NO transduces its effects by reacting either directly with heme and non-heme centers of proteins or indirectly via further oxidation to various reactive nitrogen species (RNS).5439,9429} Nitrosylation refers to the binding of an NO group to a transition metal, as typified in the activation of soluble guanylate cyclase, or to a thiol (SH) group of protein cysteine residues resulting in the formation of an S-NO moiety. This latter reaction, termed S-NO, is mediated by reactive nitrogen species of higher oxidation states of NO, such as NO2 and N2O3. S-Nitrosylation is a reversible, and seemingly specific, post-translational modification that regulates the activity of a large number of targets, including metabolic, structural, cytoskeletal, and signaling proteins. Cayman's S-Nitrosylated Protein Detection Assay employs a modification of the Jaffrey et al. 'Biotin-switch' method to allow for the direct visualization of S-nitrosylated proteins in whole cells or tissues, as well as by western blot analysis. Using this method, free SH groups are first blocked (an addition of 125.1 amu per residue) and any S-NO bonds present in the sample are then cleaved. Biotinylation of the newly formed SH groups (an addition of 523.6 amu per residue) provides the basis for visualization using streptavidin-based colorimetric or fluorescence detection. |