Free Sulfhydryl Determination
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.
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.
Prepare a "Working Solution" by mixing:
- 8.4 mLs Water
- 1 mL 1M Tris-Cl pH 8.0
- 0.5 mL DTNB solution
Make serial dilutions of DTT (or whatever control you are using)with the highest concentration about 5 mM (or 10 mM for single thiol compound).
Aliquot Working Solution into tubes (495 uL is usually fine).
Add 5 uL of the DTT solutions to each