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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. In summary, some but not all crystals freeze 'better' in liquid propane than in nitrogen. 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 container. | |||
Here a short protocol how to prepare liquid propane and how to use it. | |||
* Cryogenic burns are painful. E.g. wearing a combination of cotton gloves and latex gloves protects from liquid propane and are not too bulky for crystal handling. | |||
* 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. This might take a few minutes. | |||
* Fill the liquid propane into vials. For short time storage and crystal handling 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 it stand for a while until it is liquid again and put it pack into the nitrogen bath. | |||
* Plunge the crystals just into the vials and wait until the propane is solid. | |||
== Salts as precipitants ... == | |||
Some salts may also serve as cryoprotectants: malonate (see Acta Cryst D59, 2356), formate, ammonium sulfate (at >3.5 M), lithium sulfate, and perhaps others. Anything with a flat solubility vs temperature is a good candidate. Mixing of different cryos can often have a superior protective effect to single-component cryos of the same total concentration (the "confusion principle"), so there are a lot of combinations to try. | |||
== See also == | |||
* Cryoprotectant database used to be at http://idb.exst.jaxa.jp/db_data/protein/search-e.php? or http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html ; still to be found at http://web.archive.org/web/20111011202903/http://idb.exst.jaxa.jp/db_data/protein/search-e.php | |||
* see concentrations given in: McFerrin and Snell, J.Appl.Cryst (2002) 35, 538 and Mitchell and Garman, J.Appl.Cryst. (1996) 29, 584 | |||
* faster freezing: a simple means (blowing away the gas layer) is described by Warkentin et al. (2006). J. Appl. Crystallogr. 39, 805. | |||
* annealing: e.g. Harp, J., Timm, D. & Bunick, G. (1998) Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography. Acta Cryst. D54, 622-8; Yeh, J. & Hol, W. (1998) A flash-annealing technique to improve diffraction limits and lower mosaicity in crystals of glycerol kinase. Acta Cryst. D54, 479-80; Kriminski, S., Caylor, C.; Nonato, M., Finkelstein, K. & Thorne, R. (2002) Flash-cooling and annealing of protein crystals. Acta Cryst. D58, 459-71 | |||
* slow cooling: Warkentin, M. & Thorne, R. E. (2009) Slow cooling of protein crystals. J Appl Cryst. 42, 944-952 | |||
* This cryocrystallography webinar lists some common cryoprotectants: http://www.rigaku.com/protein/webinar-001.html | |||