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

[Kat]

Not here, Sean.

On Mon, Aug 20, 2012 at 3:49 PM, Sean Mulligan <
smulligan at nanoimagingservices.com> wrote:

> the next comment (below) is interesting too… i wonder if it would work for
> low conc cryo samples
>
>
> On Aug 20, 2012, at 12:48 PM, Sacha De Carlo <sachadecarlo at yahoo.com>
> wrote:
>
> 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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