Hi Liuqing, I agree with what Pascal has already written and can add something from my experience.
In one case I needed to purify a protein using gelfiltration to control the exact composition of the buffer before preparing for the ion exchange. The protein was only stable in high salt buffers. Decreasing salt concentration (essential to binding to ion exchange) was only possible by rapid dilution of the protein in high-salt buffer 1:10 with low-salt buffer immediately prior to loading. Briefly, each 1 ml fraction from gelfiltration was diluted to 10 ml and immediately injected on ion-exchange resin. It was a tedious protocol, consisting in 3-4 hours preparation/load phase to get 30-ish fractions injected one by one in 10 ml steps. Any other attempt (dialysis, bulk dilution of all fraction and injection via superloop, etc...) resulted in the protein crashing out of solution. Inverting gelfiltration and Ion exchange would have meant a further dialysis or desalting step to set a defined buffer composition as the affinity column elution was performed over a linear gradient and the ionic strength of the pooled peak was prep dependent. The lengthy purification was shrunk by one day using this sequence of columns and the reproducible protocol gave consistently well-diffracting crystals. Hope this helps. Ciao, Marco 2017-11-17 13:44 GMT+00:00 Liuqing Chen <519198...@163.com>: > 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 >
