Hi Harvey, Could it be that the activity you're measuring comes from a contaminant? Did you test the other fractions from SEC or IEX?
Cheers, Alex 2011/3/16 Harvey Rodriguez <h.rodriguez.x...@gmail.com> > 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 >
