Free Sulfhydryl Determination
This is the protocol I used to determine the concentration of reduced cysteine in a purified protein. It takes advantage of the redox potential of the sulhydryl group and a coliometric reagent that turns yellow upon reaction with the sulhydryl (DTNB + SH ---> 2-nitro-5-thiobenzoic acid (yellow)). A standard curve is generated using a reactive sulfhydryl compound of known concentrations (cysteine, DTT, 2-ME, etc.) and then the amount of free cysteine determined for a solution of protein is compared to the known protein concentration. In doing so, one can determine the stochiometry of cysteine to cystine in a protein.
1 M Tris-Cl, pH 8.0
2 mM DTNB dissolved in 50 mM sodium acetate
Stock of known thiol compound (I use 100 mM DTT)
===Protein of Interest (not in a thiol-containing buffer!)