I am trying to detect free thiol in protein. But the confirmation of free thiol with DTNB should be done visibly (by colour change).I have already tested this with benzyl mercaptan and cystiene (small molecule) and I got yellow colour solution. But the same condition was not helpful in case of Protein.
Consider the sensitivity of detection. Proteins are large molecules with few Cys residues. Consider a protein with a molecular weight of 30,000 and 1 Cys residue. The concentration of Cys in a 1 mg/ml (1 g/L) sample of the protein is:
(1 g/L) x (mol/30000 g) = 33 µM. Make sure the protein concentration is high enough to detect the Cys.
In addition, some or all of the Cys residues may be buried in the protein when it is folded, and some of them may be in disulfide bonds. Cys residues in disulfide bonds will not be detected by DTNB unless the disulfide bonds are first reduced. Buried Cys residues can be exposed by denaturing the protein with 6M urea.
Consider the sensitivity of detection. Proteins are large molecules with few Cys residues. Consider a protein with a molecular weight of 30,000 and 1 Cys residue. The concentration of Cys in a 1 mg/ml (1 g/L) sample of the protein is:
(1 g/L) x (mol/30000 g) = 33 µM. Make sure the protein concentration is high enough to detect the Cys.
In addition, some or all of the Cys residues may be buried in the protein when it is folded, and some of them may be in disulfide bonds. Cys residues in disulfide bonds will not be detected by DTNB unless the disulfide bonds are first reduced. Buried Cys residues can be exposed by denaturing the protein with 6M urea.
In addition to Adam's answer, if there is an MS core facility you have access to, it is much more reliable and sensitive to derivatize free thiols with iodoacetamide and measure the shift in mass.
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