To add my two cents (three Canadian)...
Dear Dr. Winant,
You haven't stated how or when your peptide was
biotinylated. Did you do it post cleavage meaning
that any and all free amines (N-terminus, Lys, Orn,
etc.) would get hit and then you'd have a serious
change in retention time, longer that is, as there
would be more than just one biotin. Also, if you
didn't use enough to cover all sites, a mixture of
various biotinylated species would occur and you'd be
in trouble as you'll have several different peaks
depending on where in the peptide the sites of
biotinylation happened.
On the other hand (there's more fingers. Stephen
Wright.), if you biotinylated on resin, and your
ninhydrin test (assuming you ran one) was
good...meaning no residual free amine...then you
should only have one biotinylated peptide after
cleavage. Therefore, you'd only be doing a 'normal'
purification of your peptide.
Regards,
Steven R. Johnson, B.S. Chem.
Research Associate, Chemistry/Development
Biomeasure, Inc. Milford, MA, USA
--- Ziqiang Wang <ziqiang@angelfire.com> wrote:
> Dear Dr. Bloomberg:
>
> i agree with your comment. and i believe it is based
> on the same elution method as the original peptide.
> however, i observed that this 1-2 minutes difference
> may not be enough for the purification purpose for
> the biotinylated peptide if you want to use
> prep-HPLC to purify the peptide after reaction,
> because in prep-HPLC the injected sample
> concentration is usually much higher to decrease the
> time and solvent use. if i need a good separation in
> prep HPLC i usually have to extend the elution
> gradient that results in longer time and still not
> 100 percent satisfactory resolution. do you have
> any experience for an alternative way to better
> purify it with HPLC?
> thank you.
>
> ziqiang wang
>
> --
>
> On Sat, 2 Jun 2001 04:12:22
> Dr G. Bloomberg wrote:
> >
> >
> >On Fri, 1 Jun 2001, Dick Winant wrote:
> >
> >> Hi all,
> >>
> >> I wonder if someone can tell me how, in general,
> peptides behave on
> >> hplc when they are derivatized with biotin.
> >>
> >> Thanks for your help.
> >>
> >>
> >> Dick Winant
> >> PAN Facility
> >> Stanford Univ.
> >>
> >>
> >Dear Dick
> >
> >Biotinylated peptides usually elute one or two
> minutes later
> >than their non-biotinylated versions with standard
> RP solvents/gradients.
> >
> >Generally they behave as normal peptides but can be
> less soluble.
> >
> >Dr Graham Bloomberg
> >Peptide Synthesis Core Facility
> >Dept. Biochemistry
> >Medical School
> >University of Bristol
> >Bristol BS8 1TD
> >UK
> >
> >
> >
> >
>
>
> Get 250 color business cards for FREE!
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>
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This archive was generated by hypermail 2b29 : Wed Nov 21 2001 - 14:17:19 EST