breaking disulphide bonds

[Peter Pediaditakis]

In article 
<Pine.SGI.4.33.0104271142210.25940994-100000 at mole.bio.cam.ac.uk>,
 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?
> Thanks in advance for your opinions.  Mike.
> 

Trhow the BME out.  Try reducing with DTT final concentration of 10mM.  
At room temperature for 15 minutes should be more than suficient for 
reducing any disulfide bounds.  If this does not work, then those bonds 
are not disulfide

-- 
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology

remove "d-l-t" for reply