Jacob,
This is not a uncommon problem with Ni-chelation chromatography (on
Ni-NTA or Talon) with soluble and membrane proteins. In most of my
experiences, it is a salt problem (i.e., too little), but
hydrophobicity issues abound, as well.
On a pilot scale, just add any of the suggested additives (EDTA, DTT,
extra salt, or even just a different buffer) to your tubes before
taking fractions. Thus, the eluted protein goes drops into a
"better" solution environment. If you set it up right, the protein
is not exposed to high concentrations of any of the above, nor is
diluted too much. For example, for 1 mL fractions, add 0.25-0.5 mL
of buffer with the additives so the final volume per fraction is
1.25-1.5 mL.
Other things to add/change are: (1) pH, (2) a little detergent or
0.5 M LiCl (to reduce hydrophobic interactions), (3) some NDSB
compounds (non-detergent sulfobetaines; see the Anatrace or Sigma
catalogs) or L-arginine to reduce non-specific aggregation, (4) and
0.25-0.5 M trimethylaminoxide. And the list goes on. It is tedious
work, but not too difficult to do.
Good luck,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
****************************************************************
On Feb 14, 2008, at 7:26 PM, Jacob Wong wrote:
Dear all,
I just ran into this problem and would like to see if I could get
some helpful tips before my protein completely crashes out.
I have a protein as 6His fusion and it remained bound to the Ni
resin with 40 mM Imidazole wash (added to 1XPBS) but then was
eluted off with 200 mM (added to 1XPBS). The protein seemed to be
highly concentrated in the elution and began to get cloudy right
away, with more and more precipitation produced over a matter of
minutes. I felt so helpless, didn't know what to do, and then
decided to add 5% of glycerol into one of the fractions but that
made it even more cloudy (ohh no...).
While the protein is dying in the tube, do you have some quick
remedy for me? Thanks very much, -J.J.
