CNBr cleavage on PVDF then RPC column?

Dennis Templeton djt2 at po.cwru.edu
Fri Jul 15 16:58:51 EST 1994


In article <3014ntINNfef at rs1.rrz.Uni-Koeln.DE>, aweber at biolan.uni-koeln.de
(Andreas Weber) wrote:

> In article <1994Jul7.204327.24457 at oxvaxd>, warda at vax.oxford.ac.uk says:
> >
> >Using the method of Scott, Crimmins McCourt et al, we plan to CNBr 
> cleave our 
> >protein of interest on a PVDF membrane and elute off the fragments for 
> AA 
><del>
> 
> Dear Bill,
> 
> try the following protocol:
> 
> Incubate the PVDF in 7,5% BrCN (w/v) in 70% HCOOH/10%H20/20%CHCN3
> overnight under nitrogen in the dark. 1ml/per PVDF-slice.
> Remove the PVDF, dry down the solution in a speed-vac, wash two
> times with H20. Dissolve in 0.1% TFA and load on column.
> As HCOOH is a very good solvent, it should elute the peptides during
> the cleavage procedure.
> The same protocol can be adapted to BrCN-cleavage in the gel-slice,
> this minimizes loss of protein during blotting o0n PVDF.
> 

I wouldn't count on formic acid eluting your protein off of immobilon/PVDF.
many CNBr fragments are good sized and will not come off for love nor money
(though maybe for pyridine)

We routinely map proteins using the above procedure with nitrocellulose,
though even then I suspect that some peptides remain stuck to the membrane.
We then resolve them on high-conc SDS PAGE gels
-- 
Dennis J. Templeton
CWRU School of Medicine



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