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his-tag thrombin cleavage in-column? - as a purification strategy (Oct/20/2006 )

Has anybody used cleavage of a his-tag as a method to elute your protein from a Ni-NTA colum? I know that my protein will cleave with the wash buffer (10mM Imidazole .5M NaCl) in solution.
Or is it better to elute your protein, then cleave and then bind to a new Ni column?


I have done this several times and have had hit and miss luck with it. Sometimes it works very well and other times not at all. I don't know why, but thats my experience. It's worth trying though.