breaking disulphide bonds

Frank Fuerst ffrank at rz.uni-potsdam.de
Fri Apr 27 06:50:22 EST 2001


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




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