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Cryo: Difference between revisions
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(New page: == Introduction == Cryopreservation of protein crystals has at least two advantages over room temperature methods. First, it greatly reduces radiation damage of the crystallized protein, ...) |
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protocol contributed by Roger Rowlett, Colgate University Department of Chemistry | protocol contributed by Roger Rowlett, Colgate University Department of Chemistry | ||
== Freezing in liquid Propane == | |||
It is proposed that freezing in liquid propane is faster and therefore better for a protein than in liquid nitrogen. Measurements with small thermocouples by Hakan Hope [http://www.bio.net/bionet/mm/xtal-log/1995-November/002005.html]revealed that there is almost no difference in cooling rates; cooling in liquid nitrogen was even faster than in liquid propane. On the other hand Teng and Moffat [http://scripts.iucr.org/cgi-bin/paper?wb0048] showed that flash cooling in liquid propane is fastest. My experience is that some but not all crystals freeze 'better' in liquid propane than in nitrogen. I frequently lost small crystals in crystallization buffers with low surface tension, e.g. high concentrations of MPD when plunged into liquid nitrogen. Freezing in liquid propane solved the problem. Also transport of crystals embedded in solid propane is easier than in liquid nitrogen: you can take your dry-shipper with you without any liquid nitrogen left in the conatiner. | |||
Here a short protocol to prepare liquid propane and how to use it. | |||
* Cryogenic burns are painful. I wear usually a combination of cotton gloves and latex gloves. | |||
* Use pure propane | |||
* Place a metal piece (e.g. big screw nut) into a 50 ml plastic ('Falcon') tube | |||
* Put the plastic tube with metal piece into liquid nitrogen. Fix it with clamps or similar. Take care that no liquid nitrogen is in the tube. | |||
* Connect a tubing with a Pasteur pipette at the end to the valve on the propane cylinder. | |||
* Put Pasteur pipette into the plastic tube with the metal piece. Propane will condensate on the cool metal piece. | |||
* After 0.5-1 min you should have 20-30 ml of propane. | |||
* If you want to store the propane leave it in the liquid nitrogen until it is solid. Th is might take a few minutes. | |||
* I fill the liquid propane into vials. For short time storage and crystal handling I put the vials in a flat liquid nitrogen bath. If propane becomes solid after a while just remove the vial from the liquid nitrogen and put it on the desk; let is stand for a while and when it is liquid again put it pack into the nitrogen bath. | |||
* I plunge the crystals just into the vials and wait until the propane is solid. | |||