Re: [ccp4bb] Precipitating Crystallization Condition
Hi Christopher, you'll have to try to find out how they mix the components at MD and if they pH the solution afterwards or before (or not at all...) and to which pH. You can ask them; or try different mixing/pHing protocols on a small scale and see if one works in keeping all in solution. Mark Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 27 Sep 2011, at 17:55, Browning Christopher wrote: Dear All, My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow.. I'm a bit desperate as I don't have many hits for this protein. Cheers, Chris B -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] Precipitating Crystallization Condition
When you give the composition of the screen condition, you must also give the conditions of the protein buffer, since you get crystals when the two are mixed. If you analyse many small salt crystals by SDS-PAGE you may still get staining since protein will precipitate on the salt crystals. Enrico. On Tue, 27 Sep 2011 18:02:18 +0200, Mark J van Raaij mjvanra...@cnb.csic.es wrote: Hi Christopher, you'll have to try to find out how they mix the components at MD and if they pH the solution afterwards or before (or not at all...) and to which pH. You can ask them; or try different mixing/pHing protocols on a small scale and see if one works in keeping all in solution. Mark Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 27 Sep 2011, at 17:55, Browning Christopher wrote: Dear All, My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow.. I'm a bit desperate as I don't have many hits for this protein. Cheers, Chris B -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Tel: 0041 (0) 02 16 93 04 40 -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Precipitating Crystallization Condition
PS I would also try substituting imidazole for HEPES and zinc sulphate for zinc chloride and sodium sulphate. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 27 Sep 2011, at 18:02, Mark J van Raaij wrote: Hi Christopher, you'll have to try to find out how they mix the components at MD and if they pH the solution afterwards or before (or not at all...) and to which pH. You can ask them; or try different mixing/pHing protocols on a small scale and see if one works in keeping all in solution. Mark Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 27 Sep 2011, at 17:55, Browning Christopher wrote: Dear All, My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow.. I'm a bit desperate as I don't have many hits for this protein. Cheers, Chris B -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] Precipitating Crystallization Condition
Imidazole is basic. If you mix it directly with zinc salts you will get zinc hydroxide. Most screens are prepared by mixing stock solutions. In your case I would make up the screen from 1 M imidazole, pH 7.2, 1 M ZnSO4, and 50% PEG-4000. Add the PEG last. This should work. You may or may not need this specific salt or buffer to get crystals, and could swap them out or change concentration as required. 50 mM buffer may not be sufficient to control condition pH depending on the protein storage buffer composition. Roger Rowlett On Sep 27, 2011 11:56 AM, Browning Christopher christopher.brown...@epfl.ch wrote: Dear All, My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow.. I'm a bit desperate as I don't have many hits for this protein. Cheers, Chris B -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] Precipitating Crystallization Condition
The PEG could also be the problem, so you can mix your stock solutions by the method described below and end up with the same result as soon as you add the PEG. See: Frances Jurnak: Effect of chemical impurities in polyethylene glycol on macromolecular crystallization Journal of Crystal Growth Volume 76, Issue 3, 2 August 1986, Pages 577-582 Enrico. On Tue, 27 Sep 2011 18:43:09 +0200, Roger Rowlett rrowl...@colgate.edu wrote: Imidazole is basic. If you mix it directly with zinc salts you will get zinc hydroxide. Most screens are prepared by mixing stock solutions. In your case I would make up the screen from 1 M imidazole, pH 7.2, 1 M ZnSO4, and 50% PEG-4000. Add the PEG last. This should work. You may or may not need this specific salt or buffer to get crystals, and could swap them out or change concentration as required. 50 mM buffer may not be sufficient to control condition pH depending on the protein storage buffer composition. Roger Rowlett On Sep 27, 2011 11:56 AM, Browning Christopher christopher.brown...@epfl.ch wrote: Dear All, My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow.. I'm a bit desperate as I don't have many hits for this protein. Cheers, Chris B -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Tel: 0041 (0) 02 16 93 04 40 -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Precipitating Crystallization Condition
Thanks for the replies. I think I got it figured out. The pH seems to be the definitive factor. Indeed, the more alkaline the pH, the higher the amount of precipitation. I just played a bit around with the pH of Imidazole and it's cleared up. It also seemed to help to add things in this order. Imidazole---water---ZnSO4---PEG. Cheers, Chris On Tue, 2011-09-27 at 19:15 +0200, Enrico Stura wrote: The PEG could also be the problem, so you can mix your stock solutions by the method described below and end up with the same result as soon as you add the PEG. See: Frances Jurnak: Effect of chemical impurities in polyethylene glycol on macromolecular crystallization Journal of Crystal Growth Volume 76, Issue 3, 2 August 1986, Pages 577-582 Enrico. On Tue, 27 Sep 2011 18:43:09 +0200, Roger Rowlett rrowl...@colgate.edu wrote: Imidazole is basic. If you mix it directly with zinc salts you will get zinc hydroxide. Most screens are prepared by mixing stock solutions. In your case I would make up the screen from 1 M imidazole, pH 7.2, 1 M ZnSO4, and 50% PEG-4000. Add the PEG last. This should work. You may or may not need this specific salt or buffer to get crystals, and could swap them out or change concentration as required. 50 mM buffer may not be sufficient to control condition pH depending on the protein storage buffer composition. Roger Rowlett On Sep 27, 2011 11:56 AM, Browning Christopher christopher.brown...@epfl.ch wrote: Dear All, My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow.. I'm a bit desperate as I don't have many hits for this protein. Cheers, Chris B -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Tel: 0041 (0) 02 16 93 04 40 -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
