James,
we see this often when we are purifying individual proteins from highly
concentrated commercial preparations. We think it is due to the sudden loss
of solutes by dilution that normally keep the proteins from interacting
with each other. We usually just filter them before ion chromatography. An
alternative might be to do an HIC step first so you can add salts to the
buffer to keep them soluble.
Good Luck,
Steve Decker
James Fethiere wrote:
> Hello,
>> I was just preparing to purify a soluble kinase from the cytoplasm of
> sf9 cells but ran into a little problem. After polytron, and high speed
> centrifugation, the published method indicates that the supernatant
> (soluble fraction) should be completed with a HEPES buffer pH 7.2
> containing EDTA, and 0.02% Triton X-100 before going to an S-sepharose
> column. Well, the prepared buffer looks very clear, but as soon as I
> dilute the protein fraction with this buffer, everything becomes cloudy,
> and a light precipitate forms. I don't see any reason for this. Any
> ideas????
>> Thank you
>> James
