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

Vladimir Svetlov svetlov at oncology.wisc.edu
Wed Mar 4 12:24:33 EST 1998


In article <6d62kt$7j8$1 at nntp3.u.washington.edu>, polliwog at u.washington.edu
(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?

Yes, it is. Sometimes one's better off doing the binding in the batch
rather then on the column. Ni-NTA is an especially useful resin for such
pull-down/pull-out assays since the chelating is appeaciably resistant to
high ionic strength and denaturing agents making it possible to regulate
the sensitivity/selectivity of the assay.

Regards,
V.

-- 
Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
UW-Madison
1400 University Ave.
Madison, WI 53706



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