Free Sulfhydryl Determination

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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


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