It’s been my anecdotal experience that the phase diagram for proteins is nowhere near as uniform as is presented in most textbooks. So while in the presented diagram, I would definitely support the proposed experiment, knowing you are in that region of the phase diagram is many many many experiments.
That being said, crystallization is stochastic, so the more things you do, the better off you will be. Cheers, Chris — Christopher L. Colbert, Ph.D. Associate Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Frank von Delft <frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>> Reply-To: Frank von Delft <frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>> Date: Wednesday, July 12, 2017 at 10:54 AM To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] crystallization optimization Yes, exactly. Thanks for doing the Right Thing and posting the actual diagram. On 12/07/2017 16:26, Patrick Shaw Stewart wrote: Alun I agree Frank's point is very interesting - and he intriguingly refers us to the phase diagram. Is the point that Line A is longer than Line B ? Best wishes Patrick On 12 July 2017 at 14:40, Alun R Coker <alun.co...@ucl.ac.uk<mailto:alun.co...@ucl.ac.uk>> wrote: Hi Everyone, Franks point is really interesting. We routinely reduce the protein concentration when we see too many precipitated wells, but we never dilute the screen. Has anyone tried this? All the best, Alun On 12/07/17 08:48, Frank von Delft wrote: The point I was failing to make: reducing either protein or precipitant concentration will indeed reduce nucleation, but often won't get you bigger or more single crystals: it will just make the appearance of crystals less reliable. The way to get big single reliable crystals is to increase protein and greatly reduce precipitant. (Even better: do seeding. Like Vicky said. Incredible how often people don't bother to do seeding, yet it solves so many problems.) phx On 12/07/2017 07:50, Vicky Tsirkone wrote: Dear Frank, I may see in the attached pic several nucleation points and a considerable amount of microcrystals. Based to my knowledge decreasing the concentration of the precipitant and/or the protein concentration would be a reasonable approach to refine the initial hits. By checking the diagram as you correctly mentioned you may see that the fine tuning of protein and precipitant concetration may lead to the desirable result without reaching the precipitation zone. Patrick just check your screens. Just a rule of thumb, if you see precipitation in the ~60% of your drops then you should definitely reduce the protein concentration. ps dont forget to try the streak seeding, as well. Have a nice day and again good luck. Vicky On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft <frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>> wrote: Actually, you should try increasing the protein concentration - a lot. But be prepared to drop the precipitant concentration to almost nothing (1 or 2% isn't "low"). To understand why, look at the phase diagram and what we assume about vapour diffusion. (Which I'm assuming is what you're doing.) On 12/07/2017 06:28, Vicky Tsirkone wrote: Dear Patrick, You may reduce the protein concentation, as well. Another option could be the streak seeding by exploiting the drop of your initial condition. Good luck, V.T. On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart <patr...@douglas.co.uk<mailto:patr...@douglas.co.uk>> wrote: Microseed them into two or three random screens. Search for MMS and rMMS online. Good luck Patrick On 10 July 2017 at 15:47, Liuqing Chen <519198...@163.com<mailto:519198...@163.com>> wrote: hello everyone! I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my protein grow small needle like crystals, how can i optimize it to get bigger crystals? the attach is the crystals figure. thanks in advance sincerely Liuqing Chen -- patr...@douglas.co.uk<mailto:patr...@douglas.co.uk> Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090<tel:01488%20649090> US toll-free 1-877-225-2034<tel:%28877%29%20225-2034> Regd. England 2177994, VAT Reg. GB 480 7371 36 -- Dr Alun R. Coker Senior Lecturer Wolfson Institute for Biomedical Research University College London The Cruciform Building London WC1E 6BT Tel: 020 7679 6703 Ext 46703<tel:020%207679%206703> Web: www.ucl.ac.uk/pxmed<http://www.ucl.ac.uk/pxmed> -- patr...@douglas.co.uk<mailto:patr...@douglas.co.uk> Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090<tel:01488%20649090> US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
