Hi Jacob,
Two points which I don't think others have mentioned:
Have you checked the pH of your imidazole (or pHed the solution before
elution)? It may be that the imidazole has driven up your pH, causing
protein to crash out.
You can add various things to fraction tubes prior to elution so that
the protein gets into the appropriate buffer ASAP (e.g. more buffer for
dilution or 1M NaCl ...)
Cheers,
Charlie
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.
--
Charlie Bond
Professorial Fellow
University of Western Australia
School of Biomedical, Biomolecular and Chemical Sciences
M310
35 Stirling Highway
Crawley WA 6009
Australia
[EMAIL PROTECTED]
+61 8 6488 4406
