Subseq FPLC on H6-tag prot

[Martin Laurberg]

Hi,

I try to extensively purify a his6-tagged protein which has been purified on a  Ni2+ affinity column. On an coomassie blue stained SDS gel and also on an 
Isoelectric focusing gel I get one band and one or two very faint bands. I Wish to further purify my protein on an anion-exchange FPLC column. I elute with a linear salt gradient. 
It puzzles me that I get two tops of similar sizes, one at 200 mM NaCl and the other at 800mM NaCl....?
Can some Ni2+ ions still be attached to the His6-tag?

Thank you very much,

/Martin