Check how many His are in your protein. I had a His-tagged protein that also
wouldn't come off the nickel column except when boiled in SDS sample buffer.
Turned out there were so many His that the untagged version bound just fine (and
Dept of Microbiology wrote:
>> I have cloned GFP behind a 6X his tag to purify the protein. I am binding
> it to TALON Cobolt beads. The protein almost binds too well, in that, I am
> having a lot of trouble eluting it off. I have heard rumor of someone using
> histidine in the elution buffer to compete for the removal of the his tagged
> protein. Does anyone know of a good way to elute hard to remove his tagged
>> I appreciate the help!!