His-tagged protein won't stick to Ni column

Sai siyer at bmg.bhs.uab.edu
Wed Nov 20 11:01:27 EST 1996


In article <3292CA68.1DF9 at LMB.uni-muenchen.de>, steipe at LMB.uni-muenchen.de
wrote:

> > I've battled for well over a year with getting a His-tagged protein to bind
> > to a Ni column.
> 
> This is most unusual. Assuming that you've tested every obvious
> variation on the protocol, I would wonder whether the tag has not been
> degraded off. You could attach a common peptide epitope like myc to the
> tag (outside !) and check via Western blot whether the tag is still
> there after expression (or buy the Dianova very expensive anti-His
> monoclonal). Alternatively, there are a couple of very elegant
> elternatives, S-tag, myc-tag itself, Flag-tag or the Strep-tag. See:
> 
>      Schmidt, T. G. M. & Skerra, A. (1994). One-step affinity
> purification of bacterially
>      produced proteins by means of the "Strep tag" and immobilized
> recombinant core 
>      streptavidin. J. Chromatography A 676, 337-345. 


i had posted a reponse earlier on where i said that you would need to bind
your fusion protein with low concentrations of imidazole if you are
purifying under denaturing conditions.  I was wrong...ELUTION of fusion
proteins under denaturing conditions is what i meant, since the his-tag
(should apply to both 6H and 10his tags) is not partially hidden as it
might be if it were native and not denatured...hope this makes
sense...cheers...

-- 
Sai Iyer
siyer at bmg.bhs.uab.edu
graduate student; dept of biochemistry and molecular genetics
university of alabama at birmingham



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