Unit

UNIT 14.6 Analysis of Protein S-Nitrosylation

  1. Joan B. Mannick1,
  2. Christopher M. Schonhoff2

Published Online: 1 DEC 2006

DOI: 10.1002/0471140864.ps1406s46

Current Protocols in Protein Science

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.

Author Information

  1. 1

    University of Massachusetts Medical School, Worcester, Massachusetts

  2. 2

    Tufts Cumming School of Veterinary Medicine, North Grafton, Massachusetts

Publication History

  1. Published Online: 1 DEC 2006
  2. Published Print: NOV 2006

This is not the most recent version of the article. View current version (1 FEB 2011)

Abstract

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.

Keywords:

  • nitric oxide;
  • S-nitrosylation;
  • Saville assay;
  • chemical reduction/chemiluminescence;
  • DAN assay;
  • DAF-2 assay;
  • DAF gel assay;
  • biotin switch assay;
  • anti-SNO immunofluorescence