Free Sulfhydryl Determination

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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

tris base to pH with HCl

2 mM DTNB dissolved in 50 mM sodium acetate

Stock of known thiol compound (I use 100 mM DTT)

DTT has 2 reactive thiols per molecule, this will be corrected for later

Protein of Interest (not in a thiol-containing buffer!)

Usually several proteins samples are compare: stock solution, reduced and buffer exchanged, reduced and reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove whatever free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.

Protocol

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