Thanks a lot for all the helpful replies I got!
Here is a summary:
- increase drop size to 4+4ul if protein is not the limiting factor ->
limits skin formation
- add 10-20ul well solution directly to drop
- freeze straight from the drop
- microseeding into less conc. AmSO4 to get more crystals
- add AmSO4 free motherliquor to well solution to equilibrate drop to
lower AmSO4
- add cryo solution in 1/4 of drop volume steps to drop and freeze
directly from drop -> glucose more gentle than glycerol
- http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Cryo
- reduce evaporation from drop by mounting and freezing crystals in the
cold room
- try mineral oils -> either add small amount directly to drop or move
crystal to drop with oil first
- test diffraction of crystal at RT
- crystals could be grown in a plastic tube, which can be put directly
in the cryo stream (Yevgeniy Kalinin and Robert Thorne, Acta Cryst.
(2005). D61, 1528–1532.)
- be quick
Sabine
>Sabine Schneider wrote:
>Hi everyone,
>
>We got crystals that grew in ~3.2M ammonium sulphate and some
tris-buffer at 18dgC. Unfortunately the crystals take a while to grow
(~4-5 weeks) and so >far we only have 4-5 xtals.
>I tried to freeze the crystals, but as soon as I broke though the skin
of the drop the ammonium sulphate started to crystallise. I got the
crystals out, froze them >using sort of an artifical mother liquor with
sodium malonate as cryo and tested the diffraction. The freezing seems
to be OK and it is definitely a protein >crystal. The crystal suffered
when the ammonium sulphate in the drop started to crystallise, but
didn't seem to deteriorate anymore in the cryo. Well the >corners had
already more or less disappeared by the time I got them out of the drop...
>Since we only have a few xtals at the moment and I can't try out a lot
of things, I was wondering if anyone has experienced and solved a
similar problem? My >freezing attempt so far was in an airconditioned
room with 18dgC. I thought about higher humidity and temperature in the
room, and/or adding the cryo >directly to the drop.... Any ideas are
very much appreciated!
