Re: [ccp4bb] problems of co-crystallization of protein-DNA complex
Couple of things, Ru Heng. 1. What buffer conditions is your protein in? Is it similar to the buffer you describe as using to dissolve your DNA in? In general, you can even get away with dissolving and annealing the oligos in just Tris etc. 2. Play with buffer conditions, particularly NaCl concentrations. 3. Tweak the protein and DNA ratios. For nucleosomes, we always got white precipitate if we did not always titrate the DNA to protein ratios for every individual prep, I believe optimization of the above parameters would help with the white precipitate formation. Hope that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Aug 13, 2009, at 12:56 AM, ruheng wrote: Dear CCP4bbers, I am now working on a DNA binding protein and the purity of the protein is quite good, however the results of DLS showed that the protein aggregates terribly in quite a lot of different buffer conditions I tried and still no crystals can be obtained. So I am going to co-crystallize the protein in complex with DNA. I synthesized the oligonucleotides varying different numbers of basepairs to determine the optimal length which can bound to my protein by EMSA. I dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then annealed the DNA into the double stranded form at a final concentration of 50uM. When I performed the EMSA experiment, I mixed the purified protein with the dsDNA at the molecular ratio approximately 1:1, but white precipitate was generated as I mixed them. Does anyone have this kinds of experience when working on DNA binding proteins and co-crystallizing the protein-DNA complex? Any suggestions from yours will be appreciated. Thank you all. Ru Heng 搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！
Re: [ccp4bb] problems of co-crystallization of protein-DNA complex
Ru Heng, It is commonly helpful to combine your protein and DNA under dilute conditions and then concentrate the complex. Combining concentrated DNA and protein together has a very good chance of precipitating in my experience. I completely agree that trying different buffer conditions is a good idea (try to find one that keeps your protein happy as evaluated by DLS). Good luck, -Andy On 8/13/2009 8:23 AM, Raji Edayathumangalam wrote: Couple of things, Ru Heng. 1. What buffer conditions is your protein in? Is it similar to the buffer you describe as using to dissolve your DNA in? In general, you can even get away with dissolving and annealing the oligos in just Tris etc. 2. Play with buffer conditions, particularly NaCl concentrations. 3. Tweak the protein and DNA ratios. For nucleosomes, we always got white precipitate if we did not always titrate the DNA to protein ratios for every individual prep, I believe optimization of the above parameters would help with the white precipitate formation. Hope that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Aug 13, 2009, at 12:56 AM, ruheng wrote: Dear CCP4bbers, I am now working on a DNA binding protein and the purity of the protein is quite good, however the results of DLS showed that the protein aggregates terribly in quite a lot of different buffer conditions I tried and still no crystals can be obtained. So I am going to co-crystallize the protein in complex with DNA. I synthesized the oligonucleotides varying different numbers of basepairs to determine the optimal length which can bound to my protein by EMSA. I dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then annealed the DNA into the double stranded form at a final concentration of 50uM. When I performed the EMSA experiment, I mixed the purified protein with the dsDNA at the molecular ratio approximately 1:1, but white precipitate was generated as I mixed them. Does anyone have this kinds of experience when working on DNA binding proteins and co-crystallizing the protein-DNA complex? Any suggestions from yours will be appreciated. Thank you all. Ru Heng 搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！ http://bing.com.cn?FORM=M00HCNPubl=WLHMTAGCrea=TEXT_Search_Where_You_Are_1X1
Re: [ccp4bb] problems of co-crystallization of protein-DNA complex
Hi ruheng, Since you synthesized the oligos, you probably already know if there is any residual salt or buffer in your oligos. I don't know if that caused the problem. Sometimes people purify and desalt the oligos before the annealing step. Joe ruheng wrote: Dear CCP4bbers, I am now working ona DNA binding protein and the purity of the protein is quite good, however the results of DLS showed that the protein aggregates terribly in quite a lot of different buffer conditions I tried and still no crystals can be obtained. So I am going to co-crystallize the protein in complex with DNA. I synthesized the oligonucleotides varying differentnumbers of basepairs to determine the optimal length which can bound to my protein by EMSA. I dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and1mM DTT, pH 7.9 and then annealed the DNA into the double stranded form at a final concentration of 50uM. When I performed the EMSA experiment, I mixed the purified protein with the dsDNA at the molecular ratio approximately 1:1, but white precipitate was generated as I mixed them. Does anyone have this kinds of experience when working on DNA binding proteins and co-crystallizing theprotein-DNA complex? Any suggestions from yours will be appreciated. Thank you all. Ru Heng 搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！
[ccp4bb] problems of co-crystallization of protein-DNA complex
Dear CCP4bbers, I am now working on a DNA binding protein and the purity of the protein is quite good, however the results of DLS showed that the protein aggregates terribly in quite a lot of different buffer conditions I tried and still no crystals can be obtained. So I am going to co-crystallize the protein in complex with DNA. I synthesized the oligonucleotides varying different numbers of basepairs to determine the optimal length which can bound to my protein by EMSA. I dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then annealed the DNA into the double stranded form at a final concentration of 50uM. When I performed the EMSA experiment, I mixed the purified protein with the dsDNA at the molecular ratio approximately 1:1, but white precipitate was generated as I mixed them. Does anyone have this kinds of experience when working on DNA binding proteins and co-crystallizing the protein-DNA complex? Any suggestions from yours will be appreciated. Thank you all. Ru Heng _ 上Windows Live 中国首页，下载最新版Messenger！ http://www.windowslive.cn
