native HisTag elution alternative

Fred dynamin at hotmail.com
Tue Oct 31 22:35:24 EST 2000


I just read that L-histidine can work really well.  In theory it sounds
exactly like what you need.  The reference is at the web site I hate the
most:
http://research.bmn.com/tto/categories/record?uid=TTO.elstto00_01689525_42_t01389&node=TOC%40%40TTO%40001%40103%40001_103&tocname=Purification+methods&tocnode=TOC%40%40TTO%40category%40purification_methods

Or briefly..


soenke behrends wrote:
> 
> 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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