In-gel detection of S-nitrosated proteins using fluorescence methods. Methods Enzymol 2008;441:53-71
Date
06/17/2008Pubmed ID
18554529Pubmed Central ID
PMC2673541DOI
10.1016/S0076-6879(08)01204-4Scopus ID
2-s2.0-52049105346 (requires institutional sign-in at Scopus site) 32 CitationsAbstract
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.
Author List
Kettenhofen NJ, Wang X, Gladwin MT, Hogg NAuthor
Neil Hogg PhD Associate Dean, Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsGels
Humans
Nitrosation
Proteins
S-Nitrosothiols
Spectrometry, Fluorescence
