Change to PEG3350 ~30% for your crystallization conditions then you have a direct cryo if you get crystals. You can mount one of those five crystals and shoot it if you have an in house facility. I would try dipping it into 25% PEG4000 with whatever else is in your reservoir condition, pay attention to pH compared to your current condition. Even if you get some residual ice rings you might be able to collect decent data. Once you are done collecting/testing/cursing and it turns out not to be salt safe the crystal to prepare a seed stock. The try seeding your other plates with clear drops.
Good luck, Jürgen On Aug 14, 2012, at 1:31 PM, Harman, Christine wrote: Hi all, I need some advice on reproducing crystals that emerged in about 2 months from a screen. The background is I set up a 96-well hangdrop screen and checked the tray after 1 week and then every 2-3day for about 3 weeks. I did notice that this particular drop seemed very dynamic overtime. I stopped looking at the tray for about 2 weeks and then when I re-checked after that time I found ~5 beautiful single growing crystals. These crystals continued to grow with a few more emerging over the next week. I am not sure of the exact time the first crystals emerged, but between the time I set up the screen to when I saw the crystals it was not quite 2 months (~1 week short). I am in the process of reproducing this hit. I set up drops with the protein at the same concentration used in the screen (~5mg/mL) and at a higher concentration ~2.5X. This protein is a Fab that was complexed with peptide before setting up initial screen. The protein is in only 0.1M Tris pH8.0. The crystal condition is 13.4% PEG 8K, 2% MPD and 0.1M imidazole pH6.5. I have checked drops (~3 weeks old) and with the more concentrated protein I see some interesting crystalline like ppt similar to what I see in the drop in the screen; however, no crystals yet. The drops with the lower concentration of protein are still somewhat clear with some having crystalline like ppt. I varied the PEG 8K concentration 12.5-14% vs MPD at 2, 4, and 6%. What I need advice on is what else can I do to speed up the crystallization process. Does anyone have suggestions besides increasing the protein concentration. I have limited amounts of protein left for optimization so I was wondering if there were any additives that were better known to facilitate faster crystallization as opposed to testing everything under the sun. Any suggestions would be great :) Thanks, Christine ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
