EMSA - general question - (May/08/2013 )
I was wondering what kind of purification method should be used in order to perform an EMSA?
I have a His-tagged protein and using Nickel columns, I can purify it under native or denaturing conditions. Apparentaly, the denatured protein is also able to bind under my conditions (I do have a control DNA, where it clearly does not bind). But is this the right way to do? Or shouldn't always native conditions be chosen?
Thanks in advance!
you should purify it the way that gives you the best yield. you can refold (much of) the protein, after purifying, by dialyzing away the denaturant.
however, if your protein is soluble (not in inclusion bodies) then it would probably be better to use native conditions.