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

> > 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

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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