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
