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Hi Xiao, what you run into here is a aqueous two phase system, phosphate and PEG tend to form that, see wikipedia entry. 35% PEG 3350 is not a bad cryo, the combination with phosphate is just unfortunate. Any chance you can replace with a different salt or buffer? I don't know if adding small PEGs will help. Glycerol, MPD, sugars or oils might be helpful. Cheers, Jan -- Jan Abendroth Beryllium Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_be4.com http://www.be4.com On Oct 13, 2014, at 6:08 PM, Xiao Xiao <[log in to unmask]> wrote: > Hi Mayer and Jason, > > Thanks for your quick reply! I read from Hampton that for PEG smaller than 5K, directly increasing its concentration is a way of making cryo. But you're right, glycerol or PEG400 will be a good way and might be easier. I'll try that first. > > Thanks a lot! > > Best, > Xiao > > 2014-10-13 17:59 GMT-07:00 Mayer, Mark (NIH/NICHD) [E] <[log in to unmask]>: > Try either glycerol or PEG 400. > Increasing concentration of 'high' MW PEG is not a good strategy. > > ________________________________________ > From: Xiao Xiao [[log in to unmask]] > Sent: Monday, October 13, 2014 8:51 PM > To: [log in to unmask] > Subject: [ccp4bb] A quick question about making PEG cryo-protectant > > Hi, > > Since my crystallization condition contains PEG, I am trying to use PEG as one of my cryo for testing. For example, one crystallization condition is 0.2M Na2PO4, 20%PEG3350. I want to make 0.2M Na2PO4 and 40% PEG3350 as cryoprotectant. When I added 200ul salt from 1M stock, and 800ul PEG from 50% stock, mixed them, seems they stayed as emulsion (confirmed by microscope), no matter how I pipette or vortex them. I tried to incubate it at 40degree, seems it got worse. > > I am now trying to re-make the cryo from solid reagent, not solution stock. Will this be helpful? Or is there any other trick? > > I will really appreciate! > > Best, > Xiao >