Help! My HisTag Protein won't come off Ni Resin

Gareth gphillip at peds.bsd.uchicago.edu
Thu Feb 10 11:53:30 EST 2000


Pamela,

Could be aggregated protein that's getting left behind. Do you c'fuge/ filter
your protein and have it in the same buffer as the column before loading?
Or if you think the cysteines are causing a problem perhaps you could load in
the presence of DTT to keep them reduced.

Hope that helps

Gareth Phillips

Pamela Beckmann wrote:

> In a DENATURING (6M) urea prep of the extracellular domain of a His tagged
> protein (lots of Cys) expressed in E Coli, much of the protein stubbornly
> refuses to elute off the nickel resin.
>
> We have tried the obvious, like low pH (3), EDTA up to 100mM,  6M guanidine
> HCl,
> Triton up to 100%, and even Acetonitrile - in case the reaction was a
> hydrophobic one. In all cases there was always some bound protein left on
> the resin after treatment (as ascertained by running a small sample of the
> resin on an SDS gel and Coomassie staining).
>
> Has anyone had a similar problem? Any ideas will be welcome!
>                                                     Pamela Beckmann, NIMR
>
> ---





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