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