His6-Tagged Protein: Binding strength, panning...

Jakob Begun jb261 at cryst.bioc.cam.ac.uk
Wed Mar 4 12:20:48 EST 1998


I've pulled interacting proteins out before using a 6-His tagged protein on
Qiagne NTA-Ni beads.  To prevent non-specific interactions I used 20 mM
imidazole and 300 mM KCl in the buffer.  I found that I got the best
results when I ran the column continuously (i.e. recycling flow through)
over night and then eluting the next day.

S. Findley wrote:

> Ok I have a his6-tagged protein which I would like to immobilize on a
> Ni-NTA column and subsequently bind and purify interacting proteins by
> passing a cell extract over the column.
>
> Is this a generally used approach?
> Are there preferable tags to immobilize proteins on columns?
> (Is th affinity of His6 for the Ni sufficient for this type of protocol?
>
> Thanks
>
> SDF
> --
> Seth D. Findley               polliwog at u.washington.edu
> Department of Biochemistry    Phone: 206-543-1710
> Health Sciences Building J579
> University of Washington, Box 357350, Seattle, WA 98195



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Jake Begun
Department of Biochemistry
Cambridge University
Cambridge CB2 1QW

ph. 01223 766027
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