Hello Christine, I was working with Fab-peptide complexes and found that streak microseeding (for instance, with a cat whisker or horse-hair) was helpful for obtaining reproducible crystallization conditions - spontaneous nucleation relies on too many imponderables. However, I would also echo Yuri's, Juergen's, and Ho's advice - shoot one of the crystals you have and (a) make sure it's not just salt (unlikely given the PEG/MPD condition) and (b) see if it's actually OK without optimization.
Good luck! Best, Anna On Tue, Aug 14, 2012 at 10:31 AM, Harman, Christine <[email protected]> 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 >
