When I was a practicing chemist, I had some experience with negatively
charged peptides, and it was all quite nasty. I suggest you (a) try to
do it on reversed phase (probably, in ammonium bicarbonate/carbonate
buffer), (b) try to do ion exchange on a low pressure system, possibly
with pH gradient, and (c, but first) get sure your product loads on
the column in your conditions at all. IMHO, volatile buffers (ammonium
acetate, ammonium bicarbonate...) are more practical than Tris/NaCl.
Vladimir
On 30/11/99, Jenny Shipway wrote:
> 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
Vladimir Titov
Bokiron Ltd., Moscow, Russia
vmtitov@aha.ru