i am trying to identify cysteine residues in my protein that undergo s-glutathionlyation (oxidised glutathione linked with oxidised cysteine residues in protein via disulfide linkage; P-SSG) under oxidative stress conditions.
i used diamide as an oxidising agent following which i lyse the cells and IP by flag tagged protein and probe it with anti-glutathione antibody . i supplement my lysis buffer with alkylating agent 50mM n-ethylacetamide (NEM) to prevent disuflide bonds reforming post experimental conditions.
well i do identify s-glutathionlyation in my protein under non-reducing conditions, but as a control, adding DTT to my IP should cleave the protein-glutathione disulfide linkage which reveals the specificity of the linkage on western blot.
However adding 1mM DTT does not cleave this P-SSG linkage. i am wondering whether alkylation with NEM inhibits following reduction by DTT??
welcome basic biochemical suggestions.