STREP-Tag purification

Frank Fürst ffrank at rz.uni-potsdam.de
Tue Jun 25 05:40:00 EST 2002


Philipp Wechner <philipp.wechner at uibk.ac.at> schrieb:

> >
> >
> > It might be that the His-tag prevents it from proper refolding, but
> > perhaps you didn't try hard enough finding a suitable buffer.
> 
> I don't think that the his tag prevents propper folding - because i
> came to the same results refolding the untaged protein.
> 
> >
> >
> > What have you tried so far? What do you know about the protein - size,
> > pI, are there refolding data about homologues?
> 
> size is about 20 kDa, pI at 9.1, no data about homologues (i am sure
> about this!)
> 
> i tried different pH for refolding - it worked best with 1,2 M Tris at
> pH7.3 other buffers (MOPS, HEPES, and about 10 others) were not able
> to induce refolding

Do you really need such high buffer concentrations?

> 
> i did a lot of different buffers and i am very happy that i found the
> buffer i am using now (and it took me nearly a year).

Did you ever try additives -- this is often much more promising than
just changing the buffer. Arginine works fine form many proteins (at
conc. up to molar), glycerol helps sometimes, or moderate concentrations
of certain detergents. Do you have access to the magazine of the german
GBM in your library? There's a review article about refolding using
arginine in one of the last issues.

> I do not want to go back and do refolding experiments - i think it is
> easier to find a tag that makes it able to purify my protein under
> these conditions.

I understand this very well...

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.*]




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