Hubing,
Your problem may actually be a detergent concentration mismatch
between your mother liquor and the cryosolution. This particularly
happens with vapor diffusion setups: there is a delicate balance of
"free" detergent in the mother liquor versus the proportion of the
detergent which is bound to the protein. Dropping a xtal into the
cryosolution shocks the crystal with a bolus of extra free
detergent. Hence, and counterintuitively, you may need to reduce the
detergent concentration in the cryosolution to keep everything in
balance. Try titrating down from 1% to even as low as 0.4% in the
cryosolution. Under the conditions you are using the CMC of bOG is
suppressed below the usual 0.67% (w/v).
Also, the behavior of many of the alkyl glycoside detergents is very
temperature sensitive. So be careful about the temperature of all
the solutions you use.
Regards,
Michael
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R. Michael Garavito, Ph.D. Email: [EMAIL PROTECTED]
Biochemistry & Molecular Biology Office: (517) 355-9724
Michigan State University Lab: (517) 353-9125
East Lansing, MI 48824-1319 FAX: (517) 353-9334
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On Feb 1, 2007, at 7:49 AM, Hubing Lou wrote:
Dear everybody,
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.
The crystal looked nice and I am wondering if this is just the
problem of cryo.
Does anybody have experiences on this kind of membrane protein cryo-
protect?
Any suggestions are highly appreciated!
Hubing
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