His- binding column

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


Yusen,
	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
sarkcosyl.  
Regards, 
Herb

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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