identifying s-glutathionylation NEM Vs DTT - (Feb/19/2014 )
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.
1 mM dtt is not much for breaking s-s bonds, we use 2 mM to maintain a reduced state but it won't break bonds. you should be using 10-20 mM (or more) to break bonds.
nem will prevent s-s bonds from reforming, so you may not have bonds to break (?).
I agree with mdfenko that you should use higher concentrations of DTT. However, if you haven't removed the unreacted NEM from the protein in solution, the DTT will react with the NEM instead of reducing your protein disulfide, especially since there is a huge excess of NEM over DTT. Remove the NEM or neutralize with excess DTT or other free thiol, then add DTT.