Free Sulfhydryl Determination
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==Reagents== | ==Reagents== | ||
| - | + | ===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!) | ||
==Protocol== | ==Protocol== | ||
==Comments== | ==Comments== | ||
Revision as of 11:41, 2 March 2006
Contents |
Background
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.
Reagents
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!)
