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In addition to others' excellent suggestions, I'd like to offer more option to consider: Your crystallization set-up has some form of chemical non-equilibrium with respect to protein. It may just be my imagination but something is happening to the yellow precipitate in your drops, namely it's going away. Is it possible that you have a slow proteolysis (or something) happening to your protein, where one of the proteolytic products is crystallizing, however it's not the *final* proteolytic product so crystals are transient for as long as a 'steady state pool' of the appropriate form is above the solubility threshold (this implies equilibrium between soluble protein and crystals, which is not a huge stretch). If this is the case, I'd check your drops with MS after a few days to see if anything interesting is happening. Options include but are not limited to: proteolysis, oxidation, chemical reaction of protein, chemical reaction of buffers and/or additives, and so on :) Artem P.S. It could simply be that your protein is initially precipitated but then driven back into solution? It happens. A. On Mon, Nov 28, 2011 at 4:56 PM, Mathews, Irimpan I. < [log in to unmask]> wrote: > Hi Christine, > > Another minor point (mainly for hanging drops) not mentioned in the > messages. > > When you bring the crystallization tray from lower temperature to room > temp. there is a possibility for condensation and slowly droplets near the > protein drops gets absorbed into the protein drop. If this happens, > depending on the condition and character of the protein, the crystals > could crack or dissolve. > > In your case, condensation might have happened during your last check and > the crystal may have affected by this issue. To avoid this problem: while > taking the tray from the incubator place another tray on the top of your > protein tray and keep them outside for few mts before checking with the > microscope. > > best wishes, > Mathews > > ________________________________________ > From: CCP4 bulletin board [[log in to unmask]] On Behalf Of Harman, > Christine [[log in to unmask]] > Sent: Monday, November 28, 2011 12:43 PM > To: [log in to unmask] > Subject: Re: [ccp4bb] Disappearing crystals > > 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:[log in to unmask]] On Behalf Of > Enrico Stura > Sent: Monday, November 28, 2011 1:45 PM > To: [log in to unmask] > 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 <[log in to unmask]> 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: [log in to unmask] Fax: 33 (0)1 69 08 90 71 >