We have very good experience with cation-exchange chromatography for
peptides and I would suggest to try that. Your peptide won't be sticky then.
Try strong exchanger pH around 3, usually you get very sharp peaks.
Michael Schrader
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> -----Urspržngliche Nachricht-----
> Von: Jenny Shipway [SMTP:jennys@biols.susx.ac.uk]
> Gesendet am: Dienstag, 30. November 1999 18:26
> An: Recipients of ABRF List
> Betreff: IE HPLC
>
>
> Hello you ABRFers,
>
> Help!
>
> It's easy, I thought, just get an anion-exchange column (DEAE) and get a
> salt gradient going. Hah!
>
> I have a highly charged peptide (30aa, 4 Glu's, no positive charges) which
> is refusing to budge from the column. I've been running 10mM Tris (pH 8.5
> - I want to seperate this guy from another with pI about 7, and didn't
> want to do a pH gradient) and shoving loads of NaCl in, but nothing comes
> off. I just ran 2M NaCl for 1.5h (this is how Vydac say I should clean the
> column of any highly charged stuff) and still nothing.
>
> Is there anything I can do to avoid doing a pH gradient? (Kosmotropic
> salts?) I don't want to fill the entire HPLC system with salt crystals.
> This negative peptide has a pI of about 3, so what buffer systems would be
> good for going from pH8 to pH3? Is it okay doing a pH gradient with
> DEAE? (I read somewhere that it's much better to do salt gradients(?))
>
> Any thoughts would be appreciated!
>
> Jenny Shipway
> Centre for Biomolecular Design and Drug Development
> University of Sussex
>
>
>