CNBr cleavage on PVDF then RPC column?

warda at vax.oxford.ac.uk warda at vax.oxford.ac.uk
Thu Jul 7 20:43:27 EST 1994


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



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