Hi Jacob,
try adding a bit of edta (1mM) to remove any Ni that might come off the
column. You could also add some DTT or bME to keep cysteines reduced
(careful, add only after the edta!). In my experience the gradient did
not work very weel because the protein with have a lot of impurities.
You can also think about adding a different tag (GST is usually helpful
in keeping proteins soluble).
Hope this helps!.
Jose
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.
