breaking disulphide bonds

Dr. Artem Evdokimov eudokima at mail.ncifcrf.gov
Fri Apr 27 07:16:44 EST 2001

You might want to elaborate a little bit about your protein :)
You could try CnBr digestion - this would work well under denaturing
conditions i.e. guanidine hydrochloride with 0.1 M HCl.

In addition you could try a better reducing agent i.e. TCEP (available
from Pierce).


Frank Fuerst wrote:
> Michael Witty <mw132 at mole.bio.cam.ac.uk> wrote:
> >Dear All,
> >        I am trying to fragment a protein for peptide mapping (ie
> >N-terminal sequencing and Mass Spec), BUT the protein will not fragment.
> >I am using trypsin, chymotrypsin, elastase and V8 protease.  There are
> >disulphide binds, but I have added 14mM mercaptoethanol.  My question is:
> >what are my options for more vigourous attempts at fragmenting the
> >protein?  Am I failing (for some reason) to break up the disulphides?
> Perhaps it is so stable that it practically doesn't expose the
> disulfide bonds. Try urea or guanidinium chloride, 6M of each, I'd
> suggest, together with Mercaptoethanol.
> Frank

|Dr. Artem Evdokimov   Protein Engineering |
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