[3dem] Recalcitrant viruses

[Doryen Bubeck]

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
****************************************
Doryen Bubeck, Ph.D.
Division of Structural Biology
Wellcome Trust Centre for Human Genetics
University of Oxford
Roosevelt Drive
Oxford OX3 7BN
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---- 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):
>http://picasaweb.google.com/esanzgar/20080226?authkey=eoSXkRt-Ook
>
>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
>
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