In-gel detection of S-nitrosated proteins using fluorescence methods.
Gel-based detection of protein S-nitrosothiols has relied on the biotin-switch method. This method attempts to replace the nitroso group with a biotin label to allow detection and isolation of S-nitrosated proteins and has been used extensively in the literature. This chapter describes a modification of this method that differs from the original in two major ways. First, it uses a combination of copper ions and ascorbate to achieve selective reduction of the S-nitrosothiol. Second, it replaces the biotin label with fluorescent cyanine dyes in order to directly observe the modified proteins in-gel and perform comparative studies using difference gel electrophoresis analysis in two dimensions.
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Nicholas J
NJ
Kettenhofen
Xunde
X
Wang
Mark T
MT
Gladwin
Neil
N
Hogg
D000818:Animals; D005782:Gels; D006801:Humans; D015538:Nitrosation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D026403:S-Nitrosothiols / Q000032:analysis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
D005782:Gels; D011506:Proteins; D026403:S-Nitrosothiols