[3dem] non aqueous cryoem
Ruben Diaz-Avalos
diaz at nysbc.org
Wed Jan 27 16:50:32 PST 2010
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
Cryoelectron Microscopy Facilities Manager
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
Cell: (858)220-2183
(858)822-6718 - Office; (858)534-5846 - Fax
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