Use of His-binding resin as an affinity column

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Feb 7 09:17:11 EST 2001


pl229 at medschl.cam.ac.uk ("Patrick Lynch") wrote:
:Has anyone ever carried out, or seen a paper, about binding a 6XHis protein
:onto a Ni2+ resin and then using this resin as an affinity column?  In my
:case, I want to stick a DNA binding protein to the resin and then pass a mix
:of DNA molecules through this resin.  Ideally, I would want to elute off the
:bound DNA for analysis.
:

Yes, I did this as a first step in antibody purification. Works fine
if your protein sticks to the column really tight (eg, you can wash
the column with 20 mM imidazole indefinitely without causing
elution). A potential downside for you is that in order to suppress
ionic interactions, you'll need to keep salt higher than it is 
typical in affinity chromatography (> 300 mM). If your protein
binds DNA well at this salt, it should work. 

        - Dima






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