[3dem] Virus on Carbon

[Mark Yeager]

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.
>
>
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem


-- 

********************************************************************

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

********************************************************************

This e-mail and any files transmitted with it may contain privileged and 
confidential information and are intended solely for the use of the 
individual or entity to which they are addressed. If you are not the 
intended recipient or the person responsible for delivering the e-mail 
to the intended recipient, you are hereby notified that any 
dissemination or copying of this e-mail or any of its attachment(s) is 
strictly prohibited. If you have received this e-mail in error, please 
immediately notify the sending individual or entity by e-mail and 
permanently delete the original e-mail and attachment(s) from your 
computer system. Thank you for your cooperation.

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <https://mail.ncmir.ucsd.edu/mailman/private/3dem/attachments/20140313/799a0c01/attachment-0001.html>