Free Sulfhydryl Determination

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Revision as of 08:41, 2 March 2006 by Smoore (talk | contribs) (Reagents)
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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!)