First of all, thanks very much for those who replied to my question. For those who do not follow this thread, I was asking a cryo question about membrane protein crystals grown at 4C with 0.05-0.2M AS (ammonium sulfate) and 15-26%PEG400.
Some of you suggested directly freeze the crystal in LN from the condition of 0.2M AS, 26% PEG400. In practice, it's my experience that PEG400 <30% in cold room readily get ice. Perhaps it is due to the air flow, the humidity etc in the cold room. I've also tried 20%PEG400 plus 15% glycerol, still there's ice. So I just sticked to 35% of PEG400 when freeze crystal in cold room. However, in room temperature (20C), 30%PEG400 works fine. I've got crystal in similar conditions at 20C and tried sequential soaking by gradually increase the PEG400 from 18% to 30%. I did four steps, 18%,22%,26% then 30%, each within 3-4 seconds. The crystal diffracted to 3.6A in house. Longer soaking caused cracks. Cold room cryo conditions is still in progress. I'll post it once I get good results. Hubing Quoting Peter Adrian Meyer <[EMAIL PROTECTED]>: > 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 > > ------------------------------------------------------------------ University of St Andrews Webmail: https://webmail.st-andrews.ac.uk
