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
<[log in to unmask]> 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
>
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