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 soup. Phoebe
===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Wed, 16 Mar 2011 12:16:49 -0500 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Jacob Keller ><j-kell...@fsm.northwestern.edu>) >Subject: Re: [ccp4bb] protein lost activity after size exclusion >chromatography >To: CCP4BB@JISCMAIL.AC.UK > >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 >*******************************************
