Hi Tim,
> 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.
Well, apparently I wasn't following as closely as I could've been either:
I just notice a big change in cryo concentrations and thought of
suggesting a gradual change. I haven't ever tested PEG400 alone for
cryo-protection (usually mixed with glycerol, peg4000, or propandiol).
Pete
> 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
>>
Pete Meyer
Fu Lab
BMCB grad student
Cornell University
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