IUBio

histag elution alternative

soenke behrends behrends at plexus.uke.uni-hamburg.de
Fri Oct 27 10:14:50 EST 2000


Dear netters, 

we purify a protein containing a HisTAG with 
a Ni-NTA matrix and elute the protein with 
imidazole (250mM). Unfortunately enzyme activity
of the eluted protein is strongly inhibited by 
250mM or 150mM imidazole, whereas 50mM imidazole
lead to 50% of the activity without imidazole. 

We now want to turn to an alternative elution 
approach e.g. EDTA. Has someone made good ex-
perience with this under native conditions and 
could provide us with a protocol? 

I have also been thinking about whether one could 
use high concentrations of DTT. In the manual 
(Quiaexpressionist) they write that DTT can reduce
Nickel thereby preventing nickel from binding His-tagged
proteins. High concentrations of a reducing agents 
would be good for the protein anyway, but it is not 
mentioned as an elution strategy. 

I am not so keen on low pH for elution, since I fear 
that it will not be any good for the protein. But I 
have so far no evidence on that. 

We would really appreciate any comment or tip
Thanks for your time and help
Soenke






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