Christine, I have a couple more comments First, if those crystals come back, I would certainly try microseeding into *random *screens. You should be able to pick up new conditions that have a little more salt in them, and are therefore stable regarding the movement of water. (If you can't get the crystals back, I would try microseeding with the precipitate that you have.)
Second, it's helpful to understand that the movement of water in and out of the drop is connected to heat *flows*, not temperature. The heat flow drives the movement of moisture (in addition to the salt etc. of course). Once you understand that you can easily get rid of e.g. condensation on the tape by putting e.g. a warm book on top of the plate in the cold room (or a book from the cold room under the plate at room temp). Similarly, you can prevent dilution of your drops by thinking about heat flows. Some say that it's essential to use Blundell and Johnson. Others say only Bergfors on crystallization, Lord of the Rings or even Harry Potter can provide the necessary inspiration. Good luck Patrick Ref for microseeding: Allan D’Arcy, Frederic Villarda, May Marsh. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica section D 63 (2007), 550–554. http://www.douglas.co.uk/mms.htm On Mon, Nov 28, 2011 at 8:43 PM, Harman, Christine < christine.har...@fda.hhs.gov> wrote: > Hi, > Thank you so much for your replies. A lot of you have mentioned > fluctuations in temp as the major contributor. And a few of you have asked > for more details of my protein/buffer and set up. To my knowledge, the > tray has been kept at constant 20 C (in an incubator) with exception of > course to when I remove the tray to view the drops. It could be possible > that my inspection of the tray might have contributed to an increase in > temp, but only temporarily. I am very careful about the time the drop sees > intense light, but it is possible the temp could have changed enough to > cause this problem. Just to give a few more details. My protein (a Fab > fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium > Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. > After setting up my drop with reservoir solution I add NaCl to well to > give ~75mM NaCl to match ionic strength of protein in drop which is diluted > 1:1 with well solution. I do hope this problem is temperature. Although I > am a little sad to not be able to freeze those crystals I did see, I still > consider myself lucky to get such good result from a condition right from > the screen so there will be some definite optimization set ups with this > condition. Could I safely say though that the crystals I observed are not > salt..:) I guess that is one good thing to take from this. Any more > suggestions on optimization would be very welcomed. > > Thanks again to all you, > > Christine > > > > -----Original Message----- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Enrico Stura > Sent: Monday, November 28, 2011 1:45 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Disappearing crystals > > When advice on crystallization is needed, it is important to give details > of > the protein concentration, the buffer the protein is in as well as the > method > used to grow the crystals. > > Problem: The crystallization conditions are essentially low salt: 100mM > buffer > and only 50mM CaCl2. So the buffer that the protein is in is very > important !! > Fluctuation in the reservoir/drop environment will lead to crystals > dissolving. > > Solution: Balance the salt in the reservoir and in the protein:precipitant > drop and make sure > the temperature is kept constant. > > Since I do not have all the necessary information, the diagnosis and the > solution proposed > are likely to be wrong! > > Enrico. > > On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin <yj...@brandeis.edu> wrote: > > > On 11/28/11 12:04 PM, Harman, Christine wrote: > >> Hi All, > >> I have just noticed a very strange thing and need some help in > >> understanding it. I recently found two crystals in a condition from a > >> screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and > >> 30% PEG MME 550). The small crystals appeared after a month and > >> started to grow over the next 5 days after I first saw them (see > >> pictures attached). I just check the same drop today and now the > >> crystals are gone. So I was wondering what happened and if anyone > >> experienced this before. Any insight or advice on what to do would be > >> greatly appreciated. > >> Thanks > >> Christine > >> Small 5 days later > > Hi Christine, > > I had similar experience. In my case, another crystal showed again with > > different size a few days later. Sometimes, it seems like it is a common > > event to others as well as I heard although my case only takes about a > > week to be crystallized. I'd rather wait or just set up again or in a > > slightly different way. > > > > Wish you well. > > > > Young-Jin > > > > > -- > Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office > Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab > LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE > > http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura > http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html > e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
