[ccp4bb] protein lost activity after size exclusion chromatography
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
Re: [ccp4bb] protein lost activity after size exclusion chromatography
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 ***
Re: [ccp4bb] protein lost activity after size exclusion chromatography
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 ***
Re: [ccp4bb] protein lost activity after size exclusion chromatography
Hi Harvey, Well, knowing nothing about your protein, allow me to ruminate anyway... It sounds like you are exploring the possibility of a metal ion or other cofactor being lost. This is a reasonable first thing to check, but your buffer exchange steps should allow small cofactors (smaller than most proteins that is) to pass through your membrane and away from your protein. This suggests that your loss of activity is due to the loss of something the size of your protein. Four things come to mind right away. 1) The least exotic possibility I can think of is maybe your protein is inactive all along according to your assay (your assay could have a problem in it, I would suggest trouble shooting your assay as a first step). This could result in your relatively dirtier prep falsely reporting activity because of another protein component (i.e. an impurity) that is active according to your assay, and then lost later during your purification. 2) This next idea seems unlikely, but you asked so... Could there be another protein component missing that is necessary for activity that you don't know about? This protein would be lost during purification resulting in an inactive form or your protein. 3) Probably another red herring here... Maybe your protein is not stable without lots of other proteins around. I have personally seen proteins that go to pot at low concentrations, but are very stable at high concentrations, for which this sort of reasoning is invoked. You could try adding Arg or other amino acids to keep it folded. 4) Is your protein active in a cleaved form? I have seen kinases with competent kinase domains in the absence of regulatory domains. If you run an activity assay that included the cleaved form of your protein, and then lose this cleaved form later after purifying away the cleaved protein, it would appear that you have lost activity. The most important advice I can give you is to pay attention to what your assays are really telling you, not what you think they are telling you because of useful assumptions we all make, but what the data really reports. For example, your activity assay shows no activity, the problem could be your protein, or a component of the assay, it is a bad idea to assume the protein is the only place something could be wrong. A factual analysis will hopefully allow you to trace back what you really know and where things could be going wrong. Hope this doesn't give you too many gooses to chase, hopefully somewhere in here is a spark to help you reason yourself out of your problem. Cheers~ ~Justin Quoting Harvey Rodriguez h.rodriguez.x...@gmail.com: 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
Re: [ccp4bb] protein lost activity after size exclusion chromatography
Hi Harvey, Could it be that the activity you're measuring comes from a contaminant? Did you test the other fractions from SEC or IEX? Cheers, Alex 2011/3/16 Harvey Rodriguez h.rodriguez.x...@gmail.com 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
