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Have you tried reverse purification over the Ni-NTA column? That is the typical next step in purification after His-tag cleavage. Or did you mean to say that the impurities elute off with your cleaved protein during the reverse purification? If this is the case, you could try adding a reducing agent to help prevent unwanted interactions. You could also try a subsequent purification using ion exchange. As for on-column digestion... it is possible but comparatively inefficient. Hope this helps. ------------------------------------------------------------------------------- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -------------------------------------------------------------------------------- ** Hard work often pays of in time, but Procrastination always pays off now ** ----- Original Message ----- From: "PULSARSTRIAN" <[log in to unmask]> To: [log in to unmask] Sent: Wednesday, January 18, 2012 7:56:39 AM Subject: [ccp4bb] His Purification Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U