atutter at aim.salk.edu
Fri Aug 21 23:38:40 EST 1998
>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.
Did you significantly decrease or increase the monovalent ion concentration
when you diluted your protein fractions? This can cause some proteins to
crash out. It is also possible that your protein is not stable at that pH.
Have you predicted the pI of your protein? If so, you may be able to play
around with pH conditions without comprimising binding to the S-sepharose.
Salk Institute for Biological Studies
10010 N. Torrey Pines Rd.
La Jolla, CA 92037
email: atutter at aim.salk.edu
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