[3dem] Virus particles excluded from CryoEM holey grids holes

[Sacha De Carlo]

Hi Daniel,

to "force" biological particles in the holes I have used chloroform in the past. Soak the Qf grids in chloroform for ~30 min, discard the chloroform according to your lab safety procedures, then let the grids dry in air in a fume hood. Use them right after drying without glow discharge. 


To remove unwanted stuff from a virus sample, people used to do a cesium chloride gradient back in the days. Sucrose gradient should work too, or maybe gel filtration on a superdex column... 


Good luck !

Best,
Sacha


 
=============================
Dr. Sacha De Carlo
Sr. Res. Scientist
FEI Company


________________________________
 From: "LUQUE BUZO, DANIEL" <dluque at cnb.csic.es>
To: 3dem at ncmir.ucsd.edu 
Sent: Monday, August 20, 2012 3:42 PM
Subject: [3dem] Virus particles excluded from CryoEM holey grids holes
 

 Dear all,
We are having serious problems to obtain high quality cryo-EM holey grids of an apparently excellent virus preparation. Find enclosed several cryo-EM images taken at different magnifications of the fungal virus preparation we are working with. As you can see, virions attach preferentially to the carbon surface, whereas holes are almost empty.  We could also observe the presence of other larger structures than virions of variable size (arrowheads). We suspect that the anomalous behavior of the virus particle suspension is due to the presence of these uncharacterized structures.
 
Particle concentration is routinely tested by negative staining of serial dilutions. Although dilution 1/80 is adequate to obtain a reproducible lawn of particles by negative staining, we had to use a dilution 1/5 to lay a few particles in the holes, ~20-30 particles/hole (as shown in these images). We have used many variations in our protocols with negative results; we used clean acetone washed grids, with/without glow discharge, and Quantifoil and C-flats holey grids with the same results (even using undiluted sample). SDS-PAGE and Coomasie staining of the virus preparations showed only the viral proteins and no contaminant. We suspect that these amorphous structures are related to lipids and/or nucleic acids from the virus.
 
Have anyone had a similar virus sample with this behavior? If so, How can we remove these aggregates without altering virus particles? .
 
You can get the images from this web site:
 
http://halley.cnb.csic.es/~virus/3dem/CryoEM_problems.jpg
 
Thanks in advance!
 
Best regards,
 
--
Daniel Luque, Ph.D.
Department of Structure of Macromolecules
Centro Nacional Biotecnología/CSIC
Campus de Cantoblanco
C/ Darwin nº 3.
28049 Madrid, Spain
 
 
 
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