Q: BrCN-cleavage in a gel?

kevin.bailey at nottingham.ac.uk kevin.bailey at nottingham.ac.uk
Tue May 27 11:23:19 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.
> 
> Thanks in advance,
> 
> Peter
> 
> p.weber at bham.ac.uk

			I suspect that because CNBr cleavages are done in a relatively high
concentration of acid you will find that at the very least the subsequent
gel will run very strangely. A better bet would be to do the cleavage in
solution, lyophilise the sample (may need to do it more than once, with a
buffer change to ammonium bicarb between the two) and then take this sample
up in the gel loading bufferand run the SDS-PAGE as usual. If you know what
range of fragment sizes to expect this will help in deciding which %
acrylamide gel to get the best separation of fragments.



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