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!


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