Q: BrCN-cleavage in a gel?

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue May 27 17:15:18 EST 1997


>In article <r103-2305971526350001 at mcconvillec.can.bham.ac.uk>,
>r103 at bham.ac.uk (room 103) wrote:
>
>> Hi there,
>> 
>> I am interested in a method to cleave a protein with a molecular mass >300
>> kDa with BrCN. Enzymatic digests would lead to more than 400 fragments,
>> therefore I think BrCN might be the best choice. Although I could blot and
>> cleave the protein on PVDF-membrane, I wonder whether it wouldn't be a
>> better option to cleave it in the gel and elute the peptides. 
>> Can anybody provide me with a method/literature for cleavage in the gel?
>> Suggestions for an alternative approach would also be appreciated.

There is a serious problem with doing CNBr cleavage on proteins run in
acrylamide gels; unless the gel is carefully pre-run to remove the residual
ammonium persulfate (and other oxidizing crud) you get oxidation of the
methionines, which renders them uncleavable by CNBr.  The result is the
complete spectrum of partial fragments, even if conditions would normally
lead to complete cleavage.  My sources tell me that the approach that you
are describing, even at best, is not very efficient because getting rid of
the oxidants is not really feasible.  You should take a look at some of the
proceedings from ABRF meetings; if there is a good method, it is probably
being done at a specialized facility.

Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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