Re: [ccp4bb] DNA binding protein
You can run a few µl of your sample on a native gel or agarose gel and visualise co-purified DNA by ethidium bromide/gel red staining. If your protein is stable at high salt concentrations (and a lot of DNA-binding proteins are) you can use a high salt concentration (like 1 M) in the lysis buffer. Ion exchange is also a good way to get rid of DNA, in my experience. Lisa
[ccp4bb] DNA binding protein
Hi all, I had a simple question about DNA binding protein. Is there an easy way to detect if your heterologously expressed protein is bound to DNA post purification. Also is there an easy way to strip the protein of DNA without any damage done to the protein in doing so. I would appreciate if someone can provide any valuable tips for the same. thanks, Neeraj -- Neeraj Kapoor TPCB Graduate Fellow Sakmar Lab/ Molecular Biology Biochemistry The Rockefeller University 1230 York Avenue, RRB 510 New York, NY 10021 lab.1.212.327.8284:fax.7904 mobile: 917.535.2030 http://www.rockefeller.edu/labheads/sakmar/sakmar-lab.html
Re: [ccp4bb] DNA binding protein
Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea
Re: [ccp4bb] DNA binding protein
I had a simple question about DNA binding protein. Is there an easy way to detect if your heterologously expressed protein is bound to DNA post purification. Yes. UV absorbance. DNA absorbs UV strongly, proteins do not. DNA absorbs 260 more thn 280, the opposite is true for proteins. In fact, it's always a good idea to check 260/280 ratio because quite frequently even non-DNA binding proteins purified by a single tag affinity chromatography contain enough DNA to skew estimation of protein concentration from 280 nm absorbance by several fold (9X in one case of my personal experience). Also is there an easy way to strip the protein of DNA without any damage done to the protein in doing so. No generic solution. Greatly depends on the protein and its properties. Dima
Re: [ccp4bb] DNA binding protein
TurboNuclease digests DNA and RNA to 1-4 base long fragments. It's very useful to remove nucleic acids during protein purification and virus purification. Another benefit of using TurboNuclease at cell lysis is to significantly reduce lysate viscosity. So it reduces the lysate volume for column loading. We can make a lysate of only 1L for 300 grams of insect cells and load a Ni column without back pressure problem. We can use lower g-force for lysate clearing. We got much lower 260 reading in the Ni pool as well. The specific activity of TurboNuclease (~1.3 units per ng) is thousands of fold higher than DNaseI. So the minute quantity of TurboNuclease used has no interference with down stream applications. Crystals of many proteins have been obtained when the cells were lyzed with TurboNuclease, a few without using affinity purification. TurboNuclease is protease-free and endotoxin-free. --Chun Competing Interests Statement Accelagen is the maker of TurboNuclease. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Pascal Egea Sent: Monday, August 10, 2009 4:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] DNA binding protein Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea
Re: [ccp4bb] DNA binding protein
I'd agree with Pascal as well, and can add that if you have a protein for which you use an affinity tag, often high salt (500mM) will strip the DNA off your protein, and might even keep it stable. Cheers - Miles On Aug 10, 2009, at 3:59 PM, Pascal Egea wrote: Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea Miles Pufall Postdoctoral Scholar Yamamoto Lab UC San Francisco Cellular and Molecular Pharmacology Mail Stop 2280 600 16th Street, Genentech Hall S-574 San Francisco, California 94158-2517 (415)476-4480
