Nat, A few years ago I had K channel crystals that formed under similar conditions. I found that using MPD as a cryo-protectant worked best. As for the evaporation issue, I had a little extra time as I was performing the mounting in a cold room.
Scott Pegan S, Arrabit C, Zhou W, Kwiatkowski W, Collins A, Slesinger PA, Choe S. Nat Neurosci. 2005 Mar;8(3):279-87. Epub 2005 Feb 20. On Fri, Apr 9, 2010 at 9:59 AM, James Holton <jmhol...@lbl.gov> wrote: > Yes, oil is great, but you have to be careful to choose an oil in which the > alcohol is not soluble, or the oil will suck it out of your drop, (just like > air). This is particularly annoying with detergents, which are almost all > soluble in oil. I've always thought that maybe some synthetic motor oils > (which your auto mechanic will tell you are immiscible with petroleum-based > oils) might be a good thing to try with membrane proteins. > > It is a common trick, however, to pre-saturate the oil by vortexing it > with an excess of the reservoir solution before applying it to the drop. > Obviously, however, this trick can get expensive when working with > detergents... > > -James Holton > MAD Scientist > > > Nathaniel Clark wrote: > >> I have wondered if placing a layer of oil over the drop would help >> solve the problem of the crystals moving around. Haven't tried it, >> but don't people harvest from a microbatch tray by dragging the loop >> and crystal through oil? >> >> Nat >> >> On Fri, Apr 9, 2010 at 11:21 AM, James Holton <jmhol...@lbl.gov> wrote: >> >> >>> Yes, isopropanol is a cryoprotectant, and a relatively good one. So are >>> the >>> other alcohols. It was even popular in the "olden days" when we would >>> typically set up drops that were 5-10 microliters in volume (each!). >>> These >>> take a while (minutes) to evaporate, giving you enough working time to >>> mount >>> the crystal before the alcohol concentration changed "too much". Modern >>> nanoliter-scale drops have largely made alcohol additives impractical, >>> which >>> is a shame. >>> >>> A potentially general way to deal with evaporating drops is to bathe the >>> work area in a stream of air or nitrogen that has been pre-saturated with >>> the reservoir solution. That is, run the gas line in and out of a jar of >>> say about 50-100 mL of replicated reservoir solution (bubbling the gas >>> through the solution in the jar) and then route the end of the hose to >>> under >>> your dissecting microscope and point it at your crystallization well just >>> before you crack it open. This should give you a nice, long working >>> time, >>> and similar devices have already been reported in the literature: >>> >>> http://dx.doi.org/10.1107/S0021889801020702 >>> >>> That, or you can try to just work really quickly! >>> >>> -James Holton >>> MAD Scientist >>> >>> Chris Meier wrote: >>> >>> >>>> Dear all, >>>> >>>> I have a protein which crystallizes in 25% isopropanol, at pH4.5. >>>> >>>> Does anyone have experience freezing crystals grown in such a condition? >>>> What cryoprotectants should I try? Can isopropanol itself act as a >>>> cryoprotectant? Any suggestions on how to deal with isopropanol >>>> evaporation >>>> during mounting? >>>> >>>> Many thanks and best wishes, >>>> Chris >>>> >>>> >>>> >>>> >>>> >>> -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry & Biochemistry University of Denver
