Free Sulfhydryl Determination

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(Reagents)
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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!)

Protocol

Comments

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