philipp.wechner at uibk.ac.at
Mon Jun 24 11:01:48 EST 2002
Artem Evdokimov schrieb:
> AFAIK, it does not mind DTT. Can you use TCEP instead of DTT ? Also keep in
> mind, that up to 5-10 mM BME are not necessarily bad for Ni-NTA, at least in
> several cases we've tried.
The DTT is not the biggest problem. I want to extract my protein from inclusion
bodies in E.coli. After treating the IB with 7M Urea or 5 M GuHCl i tried to
get my protein by NTA-Chormatography (C-terminal 6xHis tag). However, the
protein does not bind too good - but its OK.
After extracting the protein i tried to renaturate it by dillution. The only
buffer that worked for this is 1,2 M Tris (not accepatable for NTA) 10 mM DTT
(also not too good for the column :)
When i try to let the renaturated protein over the column all the Nickel is
reduced (column gets brown).
therefore i am looking for a better system that is not affected by the
redox-system of the buffer
> "Philipp Wechner" <philipp.wechner at uibk.ac.at> wrote in message
> news:3D0D9528.C39B613D at uibk.ac.at...
> > I do not want to crystallize my protein - i just want to purify it. I have
> > problems with the 6His-Tag - the NTA-collumn is not compatible with the
> > i have to use (DTT has to be present, high Tris).
> > How are the yields of purified protein with the Strep Tag, does ist
> > DTT?
> > Thanks for your help
> > Artem Evdokimov schrieb:
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