UNIT 14.6 Analysis of Protein S-Nitrosylation
Published Online: 1 DEC 2006
Copyright © 2006 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Mannick, J. B. and Schonhoff, C. M. 2006. Analysis of Protein S-Nitrosylation. Current Protocols in Protein Science. 46:14.6:14.6.1–14.6.22.
- Published Online: 1 DEC 2006
- Published Print: NOV 2006
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S-nitrosylation is the binding of an NO group to a cysteine or other thiol. Like phosphorylation, S-nitrosylation is a precisely targeted and rapidly reversible post-translational modification that serves as an on/off switch for protein function during cell signaling. However, unlike phosphorylation, S-nitrosylation of proteins occurs nonenzymatically and is mediated, at least in part, by redox-regulated chemical reactions in cells. Alterations in pH, pO2, cellular reductants, transition metals, and UV light lead to the loss and/or gain of S-NO bonds. Due to the redox-sensitive nature of the modification, analysis of protein S-nitrosylation is technically difficult, since the S-NO bond is easily disrupted during sample preparation. In addition, the level of S-nitrosylated proteins in cells approaches the limit of detection of currently available technology. Despite these technical challenges, several useful methods have been developed recently to measure protein S-nitrosylation in biological samples, and these are described in this unit.
- nitric oxide;
- Saville assay;
- chemical reduction/chemiluminescence;
- DAN assay;
- DAF-2 assay;
- DAF gel assay;
- biotin switch assay;
- anti-SNO immunofluorescence