In article <35DC5C87.1CFB at mpimf-heidelberg.mpg.de>, James Fethiere
<james at mpimf-heidelberg.mpg.de> 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
I can think of several possiblilities - one being lipids in your sample.
Triton X-100 forms miceles readily with soluble lipids and this may cause your
material to get cloudy. Another reason is that Triton X-100 is a very strong
non-ionic detergent and it can cause precipitation of proteins. Try the buffer
and column without first and then try it with triton again. Those are my
suggestions and I hope they help.
David Slomczynski
