[3dem] Virus on Carbon
Mark Yeager
my3r at virginia.edu
Thu Mar 13 20:53:05 PDT 2014
Hi Luiza
We have solved this problem in the past by applying say 5 microliters of
sample to the grid, blotting with filter paper after ~90 sec, and then
applying a second 5 microliter aliquot. Then blot, plunge and freeze as
usual.
The idea is to saturate binding sites on the carbon, increasing the
propensity for the particles to reside in the holes. The concept is
similar to blocking nonspecific binding sites on nitrocellulose using
casein or BSA in performing a western blot. If sample is precious,
perhaps you could try the process with BSA, and use your sample as usual
for the final application.
Cheers
Mark
On 3/13/14 7:41 PM, Luiza Mendonça wrote:
> Dear colleagues,
>
> I work with HIV particles and (at least in my samples), they like the
> carbon film on the grids much better than the holes, but for me this
> is a big problem, as, even using high titer preps, they rarely go to
> the holes, resulting in less-than-perfect tomograms. Has anyone else
> had this problem? What did you do to solve it?
> My particles are resusspended in PBS (with gold fiducials) at the time
> of freezing.
>
> Best,
> Luiza.
>
>
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--
********************************************************************
Mark Yeager, M.D., Ph.D.
Andrew P. Somlyo Professor and Chair
Department of Molecular Physiology and Biological Physics
University of Virginia School of Medicine
andProfessor of Cardiovascular Medicine
University of Virginia Health System
Sheridan G. Snyder Translational Research Building, Rm 320
480 Ray C. Hunt Drive
Charlottesville, VA 22908
Phone: 434-243-4676
FAX: 434-243-8271
E-mail: yeager at virginia.edu
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