help! binding to nickle column

Tom Belbin tbelbin at
Tue Aug 18 07:24:29 EST 1998

In article <35D34A59.62C0 at>, liz at METHYLGENE.COM (liz) wrote:

> I am trying to bind a 180 KDa protein with a His tag to a nickle column
> (invitrogen) but most of the protein comes out in the flow through.  I
> have tried both native and denaturing conditions.  Can anybody offer any
> suggestions?

Is the tag at the N-term end?  If so, are you sure it is actually present
on the protein being expressed?

Beware of an internal start codons.  You may be getting translation
starting downstream so that the tag is not translated at all.
I speak from experience on this one.

Tom Belbin
Biochemistry Department
Memorial University of Newfoundland

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