Using Histidine as a Nickel Elution Buffer

Simon Bushell via methods%40net.bio.net (by srb10 from st-andrews.ac.uk)
Fri May 29 10:53:12 EST 2009


So I am purifying a protein complex using Ni-NTA chromatography. One of 
the subunits is his-tagged, its binding partner isn't, and they are able 
to co-purify from a nickel column.

So far so good.

However it seems that when imidazole concentrations get above 50mM, the 
complex soon comes apart. I am therefore exploring the use of histidine 
as an alternative. But I am noticing several weird behaviours that I am 
hoping someone can shine some light on. Most alarmingly, the histidine 
appears to strip the nickel from the column. At the end the resin is 
pure white, almost as if I had run EDTA over it. Can anyone please 
explain why this might happen?

This stripping might also account why my proteins elute in just 20mM 
histidine, whereas it usually elutes in 200mM imidazole. Can anyone 
comment on the relative 'potency' of histidine-mediated elution compared 
to imidazole? I would have thought they were roughly equivalent.

For the record, my binding buffer is 20mM Tris pH8.0, 150mM NaCl, 0.5% DM*

*DM is a detergent, these proteins are membrane-bound in vivo. the 
technical manual says that <1% detergent is fine and presents no problem 
when usuing imidazole for elution.

Thank you in advance

Simon



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