Re: [ccp4bb] Reservoir buffer
Theresa, In addition to the comments provided, you do need to consider the vapor diffusion experiment process as a whole. The primary reasons why we put the precipitant mixture in the reservoir, aside from being lazy, is (1) it provides a straight forward and partially accurate starting point for making artificial mother liquors for handling and soaking soaking crystals and, more importantly (2) it ensures that ALL VOLATILE components come to the expected (assumed) equilibrium values. When I speak of volatile components, I mean not only organics (isopropanol, ethanol, etc.), but also components like ammonia from ammonium sulfate. Since the reservoir volume is often ~50x greater than that of the protein drop, mass action will significantly change the concentrations of the volatile components in the protein drop, which can markedly effect even pH. I know of one group where pH drifting (due to the reservoir solution having a different pH than the drop) had them losing the crystals until they figured that out. This also underscores the importance of having your students and tech keep good notebooks even for buffer making. While we have tried to use different concentrations of a standard PEG solution or LiCl solution as reservoirs, it turned out that we had to adapt it individually to any condition with a volatile component in the protein drop. In the end, inertia and laziness ended up with us returning to the old method. You may have more will power, but you also need to ensure reproducibility. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On 13/11/2012 07:03, Theresa Hsu wrote: Dear all In *initial screening* using vapor diffusion crystallization, does it matter whether the reservoir buffer is also the precipitant in the drop or just a high salt solution like 5 M NaCl? Thank you. Theresa
[ccp4bb] Reservoir buffer
Dear all In *initial screening* using vapor diffusion crystallization, does it matter whether the reservoir buffer is also the precipitant in the drop or just a high salt solution like 5 M NaCl? Thank you. Theresa
Re: [ccp4bb] Reservoir buffer
You should and can try, but I guess 5 M NaCl will dry your drop quite fast, unless in your drop NaCl concentration is ~2,5 M :-) That is why first mixture of well and protein solution is 1:1 v/v. Other variations such as 1:9, 9:1 etc sometimes help. 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 Nov 13, 2012, at 08:03 , Theresa Hsu theresah...@live.com wrote: Dear all In *initial screening* using vapor diffusion crystallization, does it matter whether the reservoir buffer is also the precipitant in the drop or just a high salt solution like 5 M NaCl? Thank you. Theresa
Re: [ccp4bb] Reservoir buffer
The following could be of use: Newman, J. Expanding screening space through the use of alternative reservoirs in vapor diffusion (2005) Acta Cryst D61(4), 490-493 Cheers, tom From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Felix Frolow Sent: Tuesday, 13 November 2012 5:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Reservoir buffer You should and can try, but I guess 5 M NaCl will dry your drop quite fast, unless in your drop NaCl concentration is ~2,5 M :-) That is why first mixture of well and protein solution is 1:1 v/v. Other variations such as 1:9, 9:1 etc sometimes help. 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.ilmailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 13, 2012, at 08:03 , Theresa Hsu theresah...@live.commailto:theresah...@live.com wrote: Dear all In *initial screening* using vapor diffusion crystallization, does it matter whether the reservoir buffer is also the precipitant in the drop or just a high salt solution like 5 M NaCl? Thank you. Theresa
Re: [ccp4bb] Reservoir buffer
Dear Theresa, an interesting question, I think the answer is yes it does, very much. Using Raoult's law we can calculate the equilibrium vapour pressure above a 5M NaCl solution, it is a relative humidity of 82.6%. This is much lower than say a solution of 20% PEG 4000, here we can again use Raoult's law but add in a term to cope with large polymers and we get a equilibrium relative humidity of 99.3%. So you can see that you would be dehydrating your drop significantly. However, what you can see is that for most PEG solutions used in initial screening (over 1000Da at a concentration of 10-30%) the relative humidity will be in the 99% range - you could therefore use water in the reservoir for these experiments. I have made an online calculator using Raoult's law to calculate the relative humidity equilibria for precipitant solutions which can be accessed here: http://go.esrf.eu/RH Cheers, Matt On 13/11/2012 07:03, Theresa Hsu wrote: Dear all In *initial screening* using vapor diffusion crystallization, does it matter whether the reservoir buffer is also the precipitant in the drop or just a high salt solution like 5 M NaCl? Thank you. Theresa -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
