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) 353-9334 Email: 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
