His- binding column

Herbert H. Winkler herbertw at SUNGCG.USOUTHAL.EDU
Sat Jul 13 10:02:59 EST 1996

	Are you sure that the hexa-his is still on your protein?  Maybe it
was processed at the N-terminus.  Also, if you are identifying your
protein by western blot and solubilizing it in detergent, try changing the
detergent (or use S35-met).  We have been unable to get western blots to
come out in octylglucoside plus imidazole, but it is fine in SDS or

On 12 Jul 1996 ez005679 at dale.ucdavis.edu wrote:

> I have a problem of purifying over expressed protein using His-binding 
> resins provided by Novagene company.  The problem is my protein does not 
> bind to the column, even after I tried urea denaturation.   The protein I 
> am trying to purify is a memebrane binding protein and was shown to be 
> present in the binding  and washing solutions after loading, but 
> not in the eluting solution.    Could anybody please give me some advice 
> on this column purification?   Thanks in advance
> Yusen 

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