When collecting cell lysates for a Western blot, how do I induce di-sulfide bonds?

Question

I would like to conduct a simple dimerization experiment for some protein I'm collecting from a cultured cells. My thought is, that if I'm running a non-reducing, denaturing PAGE gel, then removing beta-mercaptoethanol/DTT from the sample buffer should be enough to allow di-sulfide bonds to form.I have seen several authors incubate the cells first with a drug called BSS that diffuses across membranes and creates protein cross-links. I may be wrong, but the use of this drug seems more appropriate when trying to follow up with an immunoprecipitation or a pull-down assay.If anyone has any experience with this, could you please enlighten me?

Answer 1

If you just want to preserve normal disulfide bonds, leaving out b-me and running a native gel should work. I personally don't have experience with what you're asking, but I have worked on redox regulation and it seems to me that regulating glutathione GSH:GSSG levels would be a reasonable target-where increasing the ratio of oxidized : reduced (GSSG:GSH) should increase disulfide bonds. (You may already know this- but just to provide background this is because GSSG is used as an electron acceptor, pulling the H+ off of the Sulfur on the cysteine and allowing formation of disulfide bonds)

Looking at this paper https://www.jbc.org/article/S0021-9258(20)66778-3/fulltext you could try changing the redox state of the cells prior to lysis? I know in mammals this is orchestrated by enzymatic machinery, so it may be much easier to do this in a cell than in a lysate.