Free Sulphydryl detection
achim at cam.org
Tue Mar 19 20:34:06 EST 1996
Michael Daws wrote:
> I have a 19-mer peptide (2200 MW) with a C-terminal cysteine which I
> purchased and received as a lyophilised powder. The company told me
> that the peptide would be greater than 95% monomer, however the results
> I have been getting would suggest that the majority of the peptide is
> disulphide bonded. Is there any nice easy colorimetric reagent I can
> use to detect free sulphydryl groups so that I can determine just how
> much of my peptide is disulphide bonded? Alternatively does anybody
> know a recipe for a gel that I could use to detect such a difference?
> Mike Daws
A gel is easy. Just run a SDS-gel, but run two samples.Treat one sample with
reducing SDS-buffer, that means it contains mercaptoethanol or DTT, the other
sample with a SDS-buffer without reducing agent.
If you are correct, the reduced sample should show only one band at 2200 Da, but
the non-reduced sample a major band at 4400 Da, and a minor one at 2200 Da.
You'll probably have to run a gradient gel or a highly crosslinked one. 2200 Da is
at the resolution limit for normal gels. If you have access to a Pharmacia
Phastsystem use the 10 - 25% gradient gel. This should work, I used it myself in
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