Methods Digest, Vol 48, Issue 10

Hi

Does anyone have some experiences to purify insoluble GST-fusion
protein? I tried to purify GST-fusion protein recently, but it looks
that the protein did not bind to the beads, I did coommassie stain and
western blotting and results show the protein was expressed. I purified
the same protein last year although the fusion protein degraded
partially. In the cell suspension 1M DTT and 10% Sarkosyl were added
before sonication, 10% Triton X-100 was added after sonication, the
final concentrations are: 0.7% Sarkosyl, 2% Triton X-100 and 5mM DTT.

Thanks a lot!

Tieqiao


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Am 13.05.2009, 22:44 Uhr, schrieb TieQiao Wu <[Only registered users see links. ].au>:


That can happen, and you have two options:
- use a differnet tag (or a different position of tag, N- vs C-terminal)
and hope that it will work
- do a conventional purification (IEC, SEC, HIC etc)