Detection of S-glutathionylated proteins by glutathione S-transferase overlay.
Oxidative and nitrosative stress lead to the S-glutathionylation of proteins and subsequent functional impairment. Glutathione S-transferase (GST) from Schistosoma japonicum was found to bind to the glutathione moiety of S-glutathionylated proteins, thus establishing a convenient method for detecting S-glutathionylated proteins by biotinylated GST. Applications of this method to proteins that were prepared from cultured cells and blotted onto a membrane exhibited numerous positive bands, which were abolished by treatment with dithiothreitol. Treatment of a cellular extract with nitrosoglutathione led to enhanced staining of the bands in a dose-dependent manner. The method was also applicable for the histochemical detection of S-glutathionylated proteins in situ. The positive staining by biotin-GST became faint in the presence of S-glutathionylated ovalbumin, suggesting that the reaction is specific to S-glutathionylated proteins. Collectively, these data indicate that the method established here is simple and useful for detecting S-glutathionylated proteins on blotted membrane and in situ.
Cheng
Guang
G
Department of Biomolecular Function, Yamagata University Graduate School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan.
Ikeda
Yoshitaka
Y
Iuchi
Yoshihito
Y
Fujii
Junichi
J
D000818:Animals; D019921:Biotinylation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D051379:Mice; D009928:Organ Specificity; D020543:Proteome / Q000378:metabolism*; D014018:Tissue Distribution
D020543:Proteome; D005982:Glutathione Transferase; D005978:Glutathione