Hi liuqing, It is more usual to finish with a sec because you control the composition of the final conditioning buffer of your sample and remove any aggregates. We personally favor the following sequence iMac - desalt - iex if necessary - sec
Desalt meaning one of those small Pd10 like column that enable the quick removal of imidazole and conditioning of your protein for either cleavage with a protease of you wish to remove tags and/or lowering of salt concentration to bind on an iex column if u wish to do one. That said . In some cases Doing a real sec straight after IMac has the advantage to remove some large molecular weight contaminants ( usually DNA) and some aggregates that are annoying. These choices depend on your target of course and the level of abundance too. Best, Pascal Egea, PHD UCLA Geffen School of Medicine On Fri, Nov 17, 2017 at 5:46 AM Liuqing Chen <519198...@163.com> wrote: > Hello everyone! > I have listened someone suggested that, first use affinity chromatography > (Ni-NTA), then use SEC (superdex200 increase), and finally used ion > exchange (monoQ), to purified protein, which will be used to > crystallization. > My question is why the monoQ used in the finally step, why not the SEC > used at the finally step? > > sincerely > Liuqing Chen > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu
