STREP-Tag purification

Philipp Wechner philipp.wechner at uibk.ac.at
Tue Jun 25 06:43:53 EST 2002



Frank Fürst schrieb:

> 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 know that Arginine is a common additive for refolding - in my case it did not
really improofe the efficiency. 1.2 M Tris lead to the best results (maybe 80%
renaturation). I tried all the detergents that are standing around in our lab
(tween, triton, sds, chaps, ctab, nonident, and even some comercial dishwashers :)

No I do not have access to this journal. I would be very thankfull if you could
send me the article - but if there is nothing in it but arginine as refolding
additive i do not think that i need it.

greetings


>
>
> > 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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