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CNBr cleavage on PVDF then RPC column?

Tom Thatcher ttha at troi.cc.rochester.edu
Tue Jul 12 08:13:13 EST 1994


In <1994Jul7.204327.24457 at oxvaxd> warda at vax.oxford.ac.uk writes:

>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?  


I have tried RP-HPLC of SDS-containing protein samples and it absolutely
did not work.  All the protein was retained on the column until I reached
a very high acetonitrile conc., at which point it all came off in about
4 fractions.  As little as 0.1% SDS will interfere (I read it somewhere).

I know a bunch of ways to remove detergents but they're all relatively
clumsy and I would expect to loose a significant portion of an already
precious sample.

Tom Thatcher
University of Rochester Cancer Center
ttha at troi.cc.rochester.edu



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