Free Sulfhydryl Determination

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(1 M Tris-Cl, pH 8.0)
(Protein of Interest (not in a thiol-containing buffer!))
Line 13: Line 13:
DTT has 2 reactive thiols per molecule, this will be corrected for later
DTT has 2 reactive thiols per molecule, this will be corrected for later
===Protein of Interest (not in a thiol-containing buffer!)===
===Protein of Interest (not in a thiol-containing buffer!)===
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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.
+
Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged.  Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.
==Protocol==
==Protocol==
==Comments==
==Comments==

Revision as of 10:47, 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

tris base to pH 8.0 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 compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.

Protocol

Comments

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