another option might be the age of the screen perhaps ? very silly suggestion/question: your pH meter is calibrated before you make your measurements right ?
If you have a nano-pH probe (anything that can be added to a 96well reservoir well) you could test what the final pH of your composition is in which you do obtain crystals and then pH the final mixture to that value with the same electrode. Jürgen On Dec 9, 2013, at 4:59 AM, Evgeny Osipov <e.m.osi...@gmail.com<mailto:e.m.osi...@gmail.com>> wrote: Hello, Nasir, What concentration of your ammonium sulphate stock solution? Do your solution remains immiscible without ammonium sulphate?May be addition of concentrated solution of ammonium sulphate to PEG MME could cause phase separation? Anyway, try to heat your solution a bit as recommended by Tim or use ammonium sulphate stock solution with low ( probably 1 to 2 M) concetration and tell us if it works 06.12.2013 20:39, Tim Gruene пишет: Dear Nazia Nasir, did you warm up the solution a little? This makes working with PEG solutions a lot easier. You may also note the Hampton do not maintain the pH: they use the buffers as indicated but this does not mean that the final solution still has pH 6.5. With your ingredients it probably is, but e.g. with high concentrations of imidazole the final pH would differ a lot. Best, Tim On 12/06/2013 02:37 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote: I have obtained crystals in Hampton Crustal Screen 2, condition 26.The condition has the following components: 1. 0.2 M ammonium sulphate 2. 0.1 M MES buffer pH 6.5 3. 30% PEG mononmethyl ether 5,000. When I try to prepare the screen in-house, I use the Hampton stocks of 3.5 M ammonium sulphate and 50% PEG mononmethyl ether 5,000. I make the MES buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH accurately. The water I use is filtered MilliQ. However, the PEG MME and the rest of the solution remains immiscible and thus, forms droplets of separated PEG when I set up crystallization. The original condition from Hampton has no such problems and gives good crystals. Can anyone let me know how to overcome the problem insolubility of PEG MME in the buffer mix? I have not faced such problems with other conditions which I prepare in house and have PEG in them. Further, it's not possible for me to use the original screen all the time. Thanks a lot! Regards -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com<mailto:e.m.osi...@gmail.com> ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
