t********m
发帖数: 2 | 1 I had the same problem once. No good way to improve it. Two pieces of
suggestion:
First, try different amount of thrombin, different incubation time and
different temperature.
Second, try different enzyme.
My problem was solved by adding a TEV cleavage site between the GST and my
target protein. TEV is a very sequence specific protease. My protein was
purified by glutathione column and eluted with TEV enzyme. It worked. | t********m
发帖数: 2 | 2 yes, 4oC o/n or 37oC 5,30,60 and 120min. If you do in column digestion, you
need more enzyme and your protein sample will be diluted, but you do not have
to remove the GST. To my experience, the efficiency is better. There is no
quick way to remove thrombin or GST. Any way if you want to have high quality
protein, you have to use chromatography or HPLC. In my case, after monoQ, I
got single band coomassie blue band on SDS-PAGE.
degraded
any
be
protein |
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