Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated!
Harvey
