Philipp Wechner <philipp.wechner at uibk.ac.at> schrieb:
> 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 :)
It might be that the His-tag prevents it from proper refolding, but
perhaps you didn't try hard enough finding a suitable buffer.
What have you tried so far? What do you know about the protein - size,
pI, are there refolding data about homologues?
Bye, Frank
--
> But I don't really see running SETI at Home as practical as Folding at Home. What,
> exactly, would be the benefit of finding intelligent aliens on the other side
> of the galaxy?
Maybe they're broadcasting the principles of protein folding... [from bionet.*]
