It depends on the accessibility of the cut site when bound to the column, but we have a lot of luck with doing an on-column TEV cut of his-tagged proteins. We load the protein on a cobalt column, mix in his-tagged TEV, and cut overnight. In the morning the cut protein just flows off, the tag and the TEV remains bound to the cobalt, and we go straight to SEC and trays.
-Nick Last On Nov 18, 2017, at 7:00 PM, CCP4BB automatic digest system <lists...@jiscmail.ac.uk> wrote: > > > > In continuation of this thread, what is the success rate of separating TEV > and MBP tag from my desired protein after TEV cleavage using Hydrophobic/Ion > exchange column? > I have also read TEV is less active in a high concentration of Imidazole, so > dialysis before adding TEV may work better but then reaction buffer will have > 0.5mM EDTA and 1mM DTT, can these affect Hydrophobic/Ion Exchange resin? > (EDTA and DTT certainly not good for Affinity resins though) > > Regards > Nishant >
