Many proteins secreted to the bacterial cell envelope contain cysteine residues that are involved in disulfide bonds. These disulfides either play a structural role, increasing protein stability, or reversibly form in the catalytic site of periplasmic oxidoreductases. Monitoring the in vivo redox state of cysteine residues, i.e., determining whether those cysteines are oxidized to a disulfide bond or not, is therefore required to fully characterize the function and structural properties of numerous periplasmic proteins. Here, we describe a reliable and rapid method based on trapping reduced cysteine residues with 4'-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), a maleimide compound. We use the Escherichia coli DsbA protein to illustrate the method, which can be applied to all envelope proteins.
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