Free Sulfhydryl Determination: Difference between revisions
| Line 7: | Line 7: | ||
===1 M Tris-Cl, pH 8.0=== | ===1 M Tris-Cl, pH 8.0=== | ||
tris base to pH with HCl | tris base to pH 8.0 with HCl | ||
===2 mM DTNB dissolved in 50 mM sodium acetate=== | ===2 mM DTNB dissolved in 50 mM sodium acetate=== | ||
===Stock of known thiol compound (I use 100 mM DTT)=== | ===Stock of known thiol compound (I use 100 mM DTT)=== | ||
Revision as of 08:46, 2 March 2006
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 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.
