Hi Lauri,

taking the risk that it is too late for me to still follow, wouldn´t the problem of adding DTT affect your experiment equally no matter which maleimide you use for your reaction? Reducing disulfide bonds is of course what we frequently use DTT for, so you could in fact have a problem. To avoid this I would use small DTT concentrations and avoid denaturating conditions as good as possible (work fast, use low temperature, ...). Since you might still not be able to avoid the denaturation, how about you determine the rate of denaturation by measuring after different time periods after adding DTT under your given conditions to see wheather or not you have to very about the reduction of disulfide bonds and if so to at least get a clue of how much of an influence it had.

AMS will not reduce disulfide bonds. The maleimide ester of AMS will just react with free thiols.

Adding DTT may reduce the disulfide bonds in your protein, if they are susceptible, if you use a large excess. You would get reaction of one sulfhydryl of DTT forming a bond with a sulfhydryl on your protein to generate a new free thiol on your protein. But the disulfide bond can reform again. So I would avoid adding DTT if the goal is to use AMS to titrate the number of free thiols you have. Typically, you can use a labelling agent to measure whatever free thiols you have, and then you use a reducing agent to reduce all the thiols of your protein, and add a labelling agent once more. That way you can get free thiol content and total thiol content to estimate the number of disulfide bonds you have on your protein.