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Separation of very hydrophobic peptides

Bernard Heymann bheymann at bragg.bio.purdue.edu
Sat Jul 23 12:17:03 EST 1994

In article <dh30-200794140754 at bc1.bc.chemie.th-darmstadt.de>,
dh30 at pop.th-darmstadt.de (Peter Weber) wrote:

> Hi, everybody!
> After BrCN-cleavage of a 140 KDa protein and lyophylisation, i tried to
> dissolve the products in 0.1% TFA + 15% Acetonitrile. However, only a
> certain part of the sample was redissolved. 100% Acetonitrile was nessecary
> to bring the rest of the peptides into solution. 
> I wonder if it is possible to separate these peptides by reversed-phase
> HPLC for they might get lost on the column (Id like to sequence them after
> separation). Has anybody out there any experience with the separation of
> very hydrophobic peptides?
> Thanks very much,
> Peter
> Peter Weber
> Institut of biochemistry
> University of Darmstadt
> Germany
> e-mail dh30 at pop.th-darmstadt.de

    I'm purifying a very hydrophobic protein extracted from E coli
membranes with detergent using a wide-pore (300 A) C4-reversed phase
column. Perhaps you could try to dissolve your peptides in detergents like
SDS or triton. Then you could purify them using a acetonitrile gradient.
However, be warned, both detergent and very hydrophobic peptides shorten
column life. I think both strips the organic coating from the column over
time, and the protein binds to the silica. An alternative is to use a
polymer-based column like the Poros series, which is both more expensive
and behaves in a different manner from the silica-based columns.

Good luck!

Bernard Heymann
bheymann at bragg.bio.purdue.edu

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