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
