Free Sulphydryl detection

[Achim Recktenwald]

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?
> Thanks.
> 
> 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 
this range.

Achim