His tagged protein won't bind

Allison noname at NOSPAM.com
Tue Sep 14 13:59:10 EST 2004


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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