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

[Allison Haggarty]

Check how many His are in your untagged protein.  I once had the same problem
- the only way to get the tagged protein off the nickel resin was by boiling
in SDS-PAGE sample bufffer.  In the course of troubleshooting I checked the
sequence of my protein.  Turned out that there was a higher than average
amount of His and the untagged protein would stick to the nickel resin just
fine (and could be eluted).

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
>
> ---