I guess it depends on what your "activity" is. Can you divulge that? Could it be that Ni is necessary?
Jacob On Wed, Mar 16, 2011 at 11:28 AM, Harvey Rodriguez <h.rodriguez.x...@gmail.com> wrote: > 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 -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *******************************************
