The biotin switch method for the detection of S-nitrosylated proteins.
Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. Here, we describe a novel method for detecting proteins that contain nitrosothiols. In this three-step procedure, nitrosylated cysteines are converted to biotinylated cysteines. Biotinylated proteins can then be detected by immunoblotting or can be purified by avidin-affinity chromatography. We include examples of the detection of S-nitrosylated proteins in brain lysates after in vitro S-nitrosylation, as well as the detection of endogenous S-nitrosothiols in selected neuronal proteins.
Jaffrey
S R
SR
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. E-mail: jaffrey@jhmi.edu
Snyder
S H
SH
D000818:Animals; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005189:False Positive Reactions; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
D011506:Proteins; D026403:S-Nitrosothiols; D001710:Biotin; D003545:Cysteine