Depending on the type of resin you should be able to use IMAC all the way up to pH 11 or so (works for Ni-NTA, not sure about HIS-SELECT or other resins). Obviously your buffer system changes (carbonate works well at 11).
Why not to elute the complex together with pure monomer and then try separation via SEC or some other means? Or flush the column with a gradient of urea or guanidine to see where the remaining partner protein elutes? Notably, if your interaction is hydrophobic in nature, high salt will strengthen it. By the sound of things, the remaining 20% is probably a part of a semi-denatured aggregate, so a detergent, organic, or chaotrope wash is likely to help. Artem On Wed, Nov 17, 2010 at 10:49 AM, Daniel Bonsor <[email protected]> wrote: > I have a His-tagged protein which I am coexpressing with it's binding > partner to prevent proteolysis. Once on the Nickel column I can remove 80% > of the partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 > overnight. However the last 20% is difficult to remove, even if I reload the > Nickel column and flush a further 2l of salt solution. I am wondering if I > can increase the pH to 9.0 or 9.5. It should not effect the binding of His > for the Nickel as the His-tag has to be deprotonated to bind, though will it > causing stripping of the Nickel? > > Thanks > > > Dan >
