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