A very small suggestion, is to retrive a small volume of protein solution closer to the crystal that is growing and find the protein concentration. I would think in your case you have too much nucleation happens at the same time that could be prevented by seeding into a lower protein concentration. Knowing the protein concentration around the crystal help you to find what protein concentration that you need to microseed into. Hope this helps.
On 11/22/06, Diana Tomchick <[EMAIL PROTECTED]> wrote:
*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** There are two very useful literature references that I recommend to people when they want to learn about seeding techniques: Stura E.A. and Wilson I.A. Analytical and Production Seeding Techniques. Methods, vol. 1, pp. 38-49 (1990). Stura E.A. Chapter 7: Seeding Techniques, in "Crystallization of Nucleic Acids and Proteins: A Practical Approach." Ed. Ducruix A. and Geige R., Oxford University Press, pp. 177-208 (1999). There's a lot of overlap between the two references; the first one is quicker to read, but the second one is more thorough and includes lots of detailed technique recommendations (though the instructions on preparing glass sitting drop well inserts for 24-well trays is a bit outdated). One thing to remember when seeding, is that the seeds may dissolve in drops that are not pre-equilibrated. I find that seeding as a technique for crystal optimization generally works best if the drops are pre-equilibrated for roughly 48 hours before the seeds are added to the drop. I have repeatedly grown crystals that diffracted to 1.4 angstroms from drops that also included precipitate. The only hassle was that sometimes it's tedious to remove the precipitate so that the crystals can be cryoprotected properly. But that's a small price to pay if you are able to grow crystals that diffract well. Diana On Nov 21, 2006, at 9:20 PM, Jenny wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Hi, > > I'm trying to get a crystal for one protein and so far i can only get > some small ones.However the crystals were grown from the > perticipate.After I set up the trays, the first several days i only > saw perticipitate, and then after 10 days, lots of tiny crystals show > up.I tried to use the additive screen, but still didn't change much.In > order to get a bigger crystal, what can I do the next step? > > I was thinking about seeding, is there any protocol for doing this?I'm > a little confused about one thing is that after I crushed the > crystals, I need to add to the pre-equilibrium solution, does this > mean, in my case, I should put the tiny crystals into the trays that I > already set up for 10 days?I'm using haning drop right now.Is it > easier to use sitting drop instead to do seeding?Any suggestions would > be appreciated. > > Thanks. > > Jenny
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