I have a his-tagged fusion protein that has an enterokinase site near the centre so when I cleave, you can't be sure which band is what in the resulting gel (both products run a similar distance). I was planning on running my Ek digestion over a Ni column and the flow through should contain my desired protein and the eluate my His-tagged terminus. However, I am unsure whether I need to exchange my buffers after the Ek digestion step as the column protocol is based on purification post E. coli cell lysis? Has anyone done this and have any pointers before I begin?!
Edited by darel_am, 27 January 2011 - 03:25 AM.