I had a similar problem - turns out the tagged protein doesn't bind to
Ni-NTA under native conditions but the denatured protein does. We
think that somehow the His tag is hidden or not easily accessible in the
native conformation. The protein purified fine in the presence of urea
but then refolding is tricky .....
Good luck!
Allison
Dr. Barry Panaretou wrote:
> We've tagged a protein with 6xHis, and expressed it in E.coli. It didn't
> bind nickel resin. We also tagged the C -terminus and again it didn't
> bond to nickel resin. Has anyone come across this before (protein is in
> supernatant after hard spin, so seems soluble--can also detrect with
> anti-his antobody on westerns, in both cases)
>> Barry
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