Depending on what the expected activity is, its worth considering the 
highly-depressing possibility that the activity seen in the impure sample was 
due to impurities:  for example, barely-visible-on-a-gel chaperones can give a 
nice ATP hydrolysis signal, and DNA ligases float about with an AMP covalently 
attached, and thus can do one round of ligation with no ATP/NAD added to the 

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723

---- Original message ----
>Date: Wed, 16 Mar 2011 12:16:49 -0500
>From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Jacob Keller 
>Subject: Re: [ccp4bb] protein lost activity after size exclusion 
>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
><> 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

Reply via email to