breaking disulphide bonds

Michael Witty mw132 at mole.bio.cam.ac.uk
Fri Apr 27 05:53:33 EST 2001

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.

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