I guess it depends on what your "activity" is. Can you divulge that?
Could it be that Ni is necessary?


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

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