Did you check whether your drops shrink or grow after setting them up? If
your calculation is correct, they shoud grow. In that case you are growing
crystals be salting-in: As the salt concentration decreases as the
drop volume increases, the solubility curve of your protein drops faster
than its concentration so that it precipitates (fortunately in form of a
crystal).
If that's the case you final state is not what you probably think it is
and you are 'shocking' the crystal with a high concentration of all the
constituents of the buffer.
Aside from this: did you try to slowly increase the concentration of the
cryo-protectant rather than transferring the crystal directly into it? You
can achieve this with a pipette by slowly adding the cryoprotectant to
the crystal. - where 'slowly' can vary between seconds and minutes or even
hours.
Good luck, Tim
I grow my crystals with vapor diffusion in 24-well format by hanging a drop
of equal volume protein and precipitant solution over the reservoir of
precipitant solution. Interestingly, when I do the math, the initial
osmolarity of my drop is greater than that of the reservoir (due to the high
NaCl in the protein solution). As far as I know, this runs against the
principles of the vapor diffusion method, as vapor will leave the reservoir
and enter the drop...
Keith
Department of Biochemistry & Molecular Pharmacology
970L Lazare Research Building
University of Massachusetts Medical School
364 Plantation Street
Worcester, MA 01605
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
