Ni column problem

Boris Steipe steipe at LMB.uni-muenchen.de
Tue Nov 12 06:34:09 EST 1996


Panomporn Panutat wrote:
> 
> I have a problem with eluting protein from Ni-column (QIAGEN) . I
> followed spin column protocol described by the manufacturers but the
> protein remains bound to the column. Does anyone have any suggestions as
> to how I can elute my protein without denaturing it.

Any of the following should elute protein, that is specifically bound:

Imidazole: washing with 300 mM should elute everything
pH: protonated Histidines will not bind, pH below 6.0 will elute.
EDTA: will wash down complexed Ni - try 5mM.

BUT: if you have followed the instructions, most likely your protein is
denatured and/or nonspecifically bound to the column. Then none of the
above is likely to work. Try 8M Urea (7M if you work in the coldroom), 1
M NaCl, 10 mM EDTA. This also will clean your column between runs.

Good luck,
Boris


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