[3dem] Recalcitrant viruses

Doryen Bubeck doryen at strubi.ox.ac.uk
Thu Feb 28 01:34:42 PST 2008

Hi Eduardo,
I had a similar problem when imaging my poliovirus particles. I found that the best thing was to apply a thin layer of carbon to the holey carbon grid, essentially making a well. I would evaporate a thin layer of carbon on mica and float the film on water. I then put the holey carbon grids on the film, carbon-side down. You can pull the whole thing up using parafilm and let it dry. Glow-discharge the grids carbon-side up but apply the sample to the noncarbon side, so that the viruses sit in the wells. The very thin layer of carbon does not produce a substantial amount of background noise, I was able to get subnanometer resolution. Also with the high symmetry of the virus you don't have to worry about preferred orientations on the grid.

Hope this helps,
Doryen Bubeck, Ph.D.
Division of Structural Biology
Wellcome Trust Centre for Human Genetics
University of Oxford
Roosevelt Drive
Oxford OX3 7BN

---- Original message ----
>Date: Wed, 27 Feb 2008 18:17:57 -0700
>From: Eduardo Sanz-Garcia <esanzgar at chem.byu.edu>  
>Subject: [3dem] Recalcitrant viruses  
>To: 3dem at ncmir.ucsd.edu
>Hi all,
>I am working with a virus that likes a lot the carbon and doesn't want 
>to go to the holes.
>Here are some pictures (the picture are not the best but it is possible 
>to see the effect):
>I have tried glow discharged and no glow discharged grids.
>I have also tried different type of buffers, virus concentration, 
>dialysis, and several applications of the virus in the grid.
>Has anyone had a similar problem?
>Does anyone have a solution for this problem?
>Thank you very, very much
>3dem mailing list
>3dem at ncmir.ucsd.edu

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