[3dem] non aqueous cryoem

[Ruben Diaz-Avalos]

Hi Norm,

As Masahide points out, glycerol per se is already troublesome. Here we have tried to see liposomes in buffers containing ~10% glycerol, and the images always looked very poor. Things improve significantly if the liposomes are centrifuged and resuspended with water, in which case they become nice and spherical. We have done this procedure just before freezing grids, and as far as I can tell, the proteins associated with the liposomes are still functional.

I can only imagine that propylene glycol and ethanol would add to the difficulties, since at a concentration of 42%, propylene glycol  already lowers the freezing point of a mixture with water to ~-22C, and then ethane might not be good enough to achieve vitrification, if at all possible.

Good luck!

Ruben.

********************************************

Ruben Diaz-Avalos, Ph.D.,
Technical Director,
New York Structural Biology Center,
89 Convent Ave. at 133rd St.,
New York, NY 10027
tel: (212)939-0660 x 529
cell:(917)515-6353

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  _____  

From: Norm Olson [mailto:nholson at ucsd.edu]
To: 3dem at ncmir.ucsd.edu
Sent: Wed, 27 Jan 2010 15:55:36 -0500
Subject: [3dem] non aqueous cryoem


  -- 
  There is a user in my facility that wants to plunge freeze liposomes 
  for cryoem.  The problem is that the solvent composition is not 
  aqueous.  It is 33% water, 11% ethanol, 14% glycerol, and 42%
  propylene glycol.    Has anyone ever tried something like this or is 
  it even possible?  My first attempts didn't work.
  
  Thanks
  
  Norm Olson
  
  
  ______________________________________________________________
  Norm Olson
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  1510 Bonner Hall
  Department of Chemistry & Biochemistry, MC-0378
  University of California San Diego
  La Jolla, CA 92093-0378
  nholson at ucsd.edu
  http://cryoem.ucsd.edu
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