[RASMB] detecting virus particles with AUC

[Arthur Rowe]

Veysel

I agree with John's opinions, and have run the same systems myself. You  
can of course scan at more than one wavelength, which enables you to  
differentiate between particles 'filled' with nucleic acid and 'empty  
particles'. EM done in parallel can be informative too, if you have  
access to same.

Just to be a bit more optimistic than John, though, about the danger of  
a cell leak:

(1) our SOPs require us to do a dummy run (buffer both channels) with  
every cell, which is then used without disassembly. We started this  
some years ago, and have never had a cell leak since

(2) at 3K to 10K rpm the chances of a window break are pretty low, in  
any case

(3) if perchance a cell does leak at speed, then the contents are  
discharged via the chamber down the throat of a diffusion pump filled  
with boiling oil vapour, so nothing should survive to get into the  
atmosphere via that route. Our local H&S people were happy to accept  
this argument. But obviously, as John says, de-toxing the cells will  
call for enquiry as to possible effects on cell components.

Arthur


On Sep 25, 2008, at 19:58, John Philo wrote:

> Veysel,
>
> Yes, this can be done, see:
> Berkowitz, S. A. and Philo, J. S. (2007). Monitoring the homogeneity of
> adenovirus preparations (a gene therapy delivery system) using  
> analytical
> ultracentrifugation.  Anal. Biochem. 362, 16-37.
>
> The speed will of course depend on the virus monomer size but also on  
> the
> extent of aggregation. Some adenovirus preparations were so aggregated  
> (like
> 1,000-10,000-mers) it was difficult to capture everything at 3,000 rpm
> (that's about as slow as the instrument wants to go). I've run  
> adenovirus
> usually at 3,000 rpm and AAV (a smaller one) at around 10,000 rpm, so  
> that
> is probably the right ballpark, but you'll probably need to do a  
> preliminary
> run to get the right speed.
>
> Hopefully this virus isn't a serious biosafety issue, but you will  
> want to
> think about the fact that if your cell leaks the vacuum pump will
> potentially vaporize virus into the room. Also some viruses may require
> detoxification with bleach, but that will destroy your aluminum  
> housings.
>
> John
>
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org  
> [mailto:rasmb-bounces at rasmb.bbri.org] On
> Behalf Of Veysel Kayser
> Sent: Thursday, September 25, 2008 10:34 AM
> To: rasmb at server1.bbri.org
> Subject: [RASMB] detecting virus particles with AUC
>
> Dear AUC experts,
>
> Is it possible to detect virus particles (aggregated and disassembled
> viruses as well monomers) in AUC? If so, what would be the recommended
> concentration and speed for spinning them? In addition, what is the
> sensitivity of AUC, e.g. can one detect monomer, dimer and smaller  
> particles
> at the same time? The virus has a bullet shape with 100nm X 200nm  
> size. Any
> input would be appreciated.
>
> Veysel
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Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156
FAX:  0115 0516157
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