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
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