CNBr cleavage on PVDF then RPC column?

Andreas Weber aweber at biolan.uni-koeln.de
Wed Jul 13 11:28:45 EST 1994


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 
>sequencing.  The fragments elute off the membrane in 2% SDS, 1% Triton 
>X-100, 50mM Tris pH 9.2-9.5, and the authors then use a second Western 
blot 
>to separate the fragments for sequencing.  Does anyone know if it's 
>possible to stick the fragments direct onto a reverse phase HPLC column 
>(Vydac 214TP) or does the detergent elution buffer preclude this?  (We 
>don't think we'll have enough material to be able to stain the fragments 
on 
>a filter, so RP-HPLC would be better).
>
>Thanks for any help,
>
>Bill Bennett
>CRC Growth Factors Group
>warda at vax.oxford.ac.uk

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.

Hope this helps

Andreas

PS: First try the procedure with a standard protein!


*************************************************************
* Andreas Weber                    Tel.: (49)-221-470-5664  *
* University of Cologne            Fax.: (49)-221-470-5181  *
* Department of Botany II                                   *
* Gyrhofstr. 15                                             *
* 50931 Cologne                                             *
* Germany                  email:aweber at biolan.uni-koeln.de *
*************************************************************



More information about the Methods mailing list