After performing protein S-acylation assays, I generally process protein samples after lysis by adding SDS-loading buffer with beta-mercaptoethanol. I was always weary of this as I thought the thiol would be a strong enough nucleophile to cleave the thioester bond. Routinely, to show thioester dependence upon addition of a fatty acid to a protein via reactive cysteines, we add hydroxylamine as this specifically cleaves thioesters, generating the amide. Generally, after addition of hydroxylamine, we allow the reaction to shake at room temperature for an hour. I find it confusing that a large excess of beta-mercaptoethanol would not also cleave the thioester bond. Any thoughts on why???