Hi,
sorry if I didn't follow this thread carefully enough and this has been
mentioned before: To my experience, 20-25% PEG400 by itself is enough for
cryo protection. Did you actually test whether you need to change anything
at all? Maybe you can just dip your crystals straight from the tray into
liquid nitrogen. You can easily test this by preparing the buffer and
freezing it without crystals and taking an image.
Cheers, Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Fri, 2 Feb 2007, Peter Adrian Meyer wrote:
Hi Hubing,
I have crystallized a membrane protein at cold room temperature (4°C).
The
protein was purified in 20mM Tris, pH8 with 1% bOG. The reservoir
solution
contains 0.1M HEPES pH7.5, 0.05-0.2M (NH4)2SO4 and 15%-26% PEG400.
The cryoprotectant was made in such a way that all the other ingredients
remained the same except for the PEG400 increased to 35%. The crystal
was
looped from the well and directly dipped into the cryo, however, the
crystal
cracked within seconds of soaking. Speedy soaking and transferring of
the
crytal into liquid N2 resulted 6Å diffraction in ESRF.
Not strictly related to membrane proteins, but one approach would be to a
series of transfers with gradually increasing percentages of PEG400
(instead of 26% -> 35% -> LN2;try 26% -> 30% (wait a while) -> 35% (wait
again) -> LN2 ). The idea is to avoid drastic changes to the crystal's
environment (similar to what Michael Garavito was talking about regarding
detergents).
You don't mention what temperature you're doing your cryo-soaking at; but
if you grew the crystal at 4 C it's probably a good idea to soak,
equilibrate and freeze at 4 C as well (you're probably doing this already
anyhow).
Good luck,
Pete
Pete Meyer
Fu Lab
BMCB grad student
Cornell University
