发信人: trissodium (Soso), 信区: Biology
标 题: Re: 求助！GST fusion protein cleavage-thrombin
发信站: Unknown Space - 未名空间 (Wed Mar 10 11:27:28 2004) WWW-POST

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.

【 在 hopetrue (沧海一声啸) 的大作中提到: 】
: What kind of temperature did you try? 4oC? My problem is also protein
degraded
: during thrombin cleavage. Also, another question is which way is better, in
: column digestion or in-solution digestion in term of efficiency? Is there
any
: quick way to remove thrombin and free GST from my proteins if digested in
: solution?
:
: 【 在 trissodium (Soso) 的大作中提到: 】
: : 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.
: :
: : 【 在 leohawk (leohawk) 的大作中提到: 】
: : : read some books on protein purification, I don't really know the exact
: : : way, but there are a lot of standard protocols, you will have to
: : : find out which one is good for your protein.
: : :
: : : 【 在 iblicf (小虎。。) 的大作中提到: 】
: : : : Thanks, I'll try it.
: : : : We're now thinking if the GST-D doesn't not fold properly, that would
be
: a
: : : : good explanation for the degradation right?
: : : : If I add Urea and cut it, how can I refold the protein after the
: cleavage?
: : : : 【 在 leohawk (leohawk) 的大作中提到: 】
: : : : : I belive you can add some detergent, like urea in your system to
: : : : : cut, thrombin will be fine in 3M(?) Urea, and denature of the
protein
: : : : : ensures accessibility of thrombin to the cutting site.
: : : : : but if you can't denature your protein or your protein is subject to
: : : : : thrombin cut, there is really no good way to solve the problem --
: : : : : there may be other methods that I don't know of though.
: : : : 4
: : : : uncleaved
: : : : improve
: : :
: : :
: :
: :
:
:


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