his-tagged protein purification from yeast

Thomas Seib hgtsei at med-rz.uni-sb.de
Fri Oct 31 06:56:30 EST 1997


In article <svetlov-ya02408000R3010971513550001 at news.doit.wisc.edu>,
svetlov at oncology.wisc.edu (Vladimir Svetlov) wrote:

> I'd greatly appreciate a protocol (or a pointer to such protocol) for
> purification of his-tagged proteins from yeast (S. cerevisiae) [both native
> and denaturing conditions] using Ni-NTA columns (Qiagen). Comments and tips
> derived from the personal experience will be appreciated even greater than
> greatly.
> Regards,
> V.

Hi Vladimir,
I am sceptic that one purification step on Ni-Agarose will be
sufficient to obtain pure protein. Personally I made the experience
that purification from bacterial extracts always works well, but
with any eukaryotic protein extracts there will be co-purifying
proteins due to the presence of His-clustered regions in the sequences.
This is true at least for native purifications.
Generally, the purification conditions need to be tested for any recombinant
protein. It is a good idea to use Co2+-Agarose from Talon (by Clontech)
instead of Ni-Agarose, since non-spefific binding is somewhat reduced.
Moreover, you can include low amounts (10-20 mM) of Imidazole in the binding
buffer to reduce binding of His-stretch-containing cellular proteins.

Hope this helps,
Matthias

-- 
Thomas Seib
Kantstr. 12
66292 Riegelsberg



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