I want to test for broken disulphide bridges in a protein. There seem to be a number of reagents which will detect a thiol (see section 4 in attached paper). However, because the context of these experiments is an aggregation study we are using pH ~ 2 (Hydrochloric acid). I'm guessing that in some cases that might cause issues. The only other reagent is NaCl (not essential).
Can someone suggest the best reagents and protocol? I don't need a precise quantification just a rough test in which I can compare two solutions and know which has more. I'm not keen on the Thioglo reagents as I don't have a huge amount of cash to blow. Bromobimane reactions?
I fully agree with Theo: derivatives that react with thiols via nucleophilic substitution reaction (bromobimane, iodoacetamides, etc) are not suitable at low pH as they require basic or neutral conditions. But reagents that use a Michael addition to alpha,beta-unsaturated carbonyls (i.e. contain –C=C-C=O fragment) can react at acidic pH. Maleimides belong to this group.
Following your advice about maleimides and for the benefit of anyone else who understands this as badly as I do, I've found n-(5-fluoresceinyl)maleimide which is available from sigma. From the attached paper seems to detect thiols with a strong, selective fluorescence enhancement. It is also a fair bit cheaper than the thioglo reagents.
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