In my case what simply worked is to put your solution with 40% glycerol on the side of the drop and wait for crystals to stop dancing. Fish them out swiping through the side were you have put your solution. Or I have used MPD as cryoprotectant and freeze them in cold room (4C) its amazing what cold air humidity can do for your drop. No problem with dancing crystals.
Michal Boniecki On Aug 23, 2018, at 11:38, James Holton <jmhol...@slac.stanford.edu<mailto:jmhol...@slac.stanford.edu>> wrote: Ahh, yes, the "dancing crystals problem". The good news is alcohols are really good cryoprotectants as well as excellent precipitants. Shame their use has fallen out of favor over the years, but I guess as drop sizes got smaller the evaporation problems got worse and worse. In my humble opinion, this is just an extreme case of a problem we all have already. Evaporation during harvesting is an insidious issue that is hard to monitor. It's not just alcohol, but water that evaporates, and some buffers are volatile too (like ammonium and acetate ions). Volatile buffers mean the pH changes over time. All this can easily lead to non-isomorphism between the first crystal you mount vs the last. An excellent review is: https://dx.doi.org/10.1107%2FS1399004714012310 It has already been suggested that you surround your harvesting environment with a wet towel (aka Kimwipe) soaked with the well solution, and this is a good idea to try and keep the local humidity (or alcohol-idity?) up. Another possibility is to make up 30-40 mL of your well solution, put that into a 50 mL conical tube and use one of those stone fish-tank aerators to bubble air or N2 gas through the appropriate solution for generating just the right "atomosphere" that your crystals are used to (see attached photo). You can then direct that gas through an additional length of hose in the general direction of your well before you crack it open. The point is to keep your crystals unaware of the fact that they are about to be harvested for as long as possible. In your case you have an excellent assay for when you have kept the harvesting environment properly controlled: the crystals will stop dancing. There are some devices on the market now, such as the "HC1" from Arinax, or the "Watershed" from MiTeGen that are a much more sophisticated way to do this, but check with the vendor before filling it with alcohol. Some seals don't like non-aqueous liquids. I expect there may be a safety concern about generating large amounts of alcohol vapor, especially isopropanol. You don't want to breathe that in. Best to keep away from open flames and work in a well-ventilated area, like the hood. Ethanol is less toxic but on a per-capita basis more dangerous. At the very least, don't drive yourself home afterwards. -James Holton MAD Scientist On 8/14/2018 1:58 PM, Thomas Krey wrote: Dear crystallization experts, We have 3D protein crystals grown from a microseed matrix screening vapor diffusion experiment in either 15% (v/v) Reagent alcohol HEPES Na pH 7.5 0.2 M MgCl2 or in 27% Isopropanol 0.18 M MgCl2 90 mM HEPES Na pH 7.5 10% Glycerol Upon opening the corresponding wells these crystals move quite a bit – presumably due to the volatility of the alcohols. Does anyone have a good suggestion to stabilize the swirling movements? Does anyone have experience, whether these conditions alone can serve as cryo-protectant (i.e., do we really have to fish, move into cryo solution and fish again)? Any suggestion or input would be highly welcome. Thank you very much in advance. Thomas Prof. Dr. Thomas Krey Hannover Medical School Institute of Virology Structural Virology Group Carl-Neuberg-Str. 1 D-30625 Hannover phone: +49 (0) 511 - 532 4308 email: krey.tho...@mh-hannover.de<mailto:krey.tho...@mh-hannover.de> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 <humidity_bubbler.jpg> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
