[ccp4bb] How to slow down crystallization? Need hep!
Hello everyone, I need your suggestion for slowing down crystallization for my proteinmy protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions
Re: [ccp4bb] How to slow down crystallization? Need hep!
On Mon, Feb 25, 2013 at 8:02 AM, lei feng spartanfeng...@hotmail.com wrote: I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Sometimes you can do this by adding a tiny amount of glycerol to your protein solution - I've seen 0.5% make the difference between awful plate clusters and nice individual crystals. I think it's also possible to use a combination of low concentrations and micro-seeding, but I've never done this personally. -Nat
Re: [ccp4bb] How to slow down crystallization? Need hep!
Feng Lei, I would personally recommend slowing down the process of nucleation either by lowering the temperature of crystallization or by utilizing Al's oil. These are two powerful ways to slow the overall kinetics of crystallization. Here is a relevant reference for you: http://scripts.iucr.org/cgi-bin/paper?he0181 Sincerely, lorenzo Lorenzo Ihsan Finci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityCollege of Life SciencesBeijing, China Date: Mon, 25 Feb 2013 11:02:03 -0500 From: spartanfeng...@hotmail.com Subject: [ccp4bb] How to slow down crystallization? Need hep! To: CCP4BB@JISCMAIL.AC.UK Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions
Re: [ccp4bb] How to slow down crystallization? Need hep!
Le 25/02/13 17:02, lei feng a écrit : Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions you can run crystallization at 4°C, or add agarose gel to your crystallization drop. -- Philippe Leone AFMB-UMR7257 Team 'Structural Immunology' Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France Tel: +33 491 82 55 81 Fax: +33 491 26 67 20 e-mail: philippe.le...@afmb.univ-mrs.fr
Re: [ccp4bb] How to slow down crystallization? Need hep!
a. Go with phase diagram gradually changing concentrations of protein, precipitant, salt concentrations and pH. Find where you are going out of crystallisation. Define region for streak seeding. b. Screen for ratios of precipitant to protein from 1:9 to 9:1. Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Feb 25, 2013, at 18:02 , lei feng spartanfeng...@hotmail.com wrote: Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions
Re: [ccp4bb] How to slow down crystallization? Need hep!
Hi Lei Fang, I cannot tell what technique you are using for crystallization. Also, many details like protein concentration, temperature of crystallization etc. are missing in your email so it's hard to guess what's going on. In any case, here are my thoughts. I used to get a shower of needles from hanging drops at 19C but a lot fewer and bigger crystals when I set up sitting drops under mineral oil at 19C. The crystals took lot longer to grow (two weeks as opposed to four days using hanging drop) so bear that in mind. My guess is that you may be able to find protocols if you just do a quick web search. There are many other ways to optimize crystal growth and size including playing with temperature of crystallization (lowering to 4C), amount of precipitant vs protein, drop size, crystallization technique, seeding by dilution etc. Hope that helps. Raji On Mon, Feb 25, 2013 at 11:02 AM, lei feng spartanfeng...@hotmail.comwrote: Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] How to slow down crystallization? Need hep!
Try using dioxane as an additive. I'd suggest adding 5- 15% dioxane into your well solution. This should inhibit nucleation resulting in fewer, larger crystals. On 25/02/13, Felix Frolow mbfro...@post.tau.ac.il wrote: a. Go with phase diagram gradually changing concentrations of protein, precipitant, salt concentrations and pH. Find where you are going out of crystallisation. Define region for streak seeding. b. Screen for ratios of precipitant to protein from 1:9 to 9:1. Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Feb 25, 2013, at 18:02 , lei feng spartanfeng...@hotmail.com wrote: Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions -- Roksana OgrodowiczDivision of Molecular Structure MRC National Institute for Medical Research The Ridgeway, Mill Hill London, NW7 1AA
