Did you try high NaCl (up to 4 M ) or high imidazole (up to 1 M) ? I am
studying a protein which ,when his-tagged, will not elute from Ni-NTA by
lowering the pH, but readily elutes using 300 mM imidazole or using DTT (20
mM). I have also heard that some proteins could elute by Ni2+ gradient. You
could take a look at the Qiagen web-site, the Qia-expressionist handbook gives
some troubleshooting tips.
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
> 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