[RASMB] determination of phage particle concentration

[John Philo]

Sabine,
 
At least some viruses are large enough that a significant fraction of the
apparent OD is due to scattering rather than true absorbance, so that would
adversely affect using absorbance to calculate the number concentration. For
adenovirus for example people usually add SDS before reading the OD to
disrupt the particles and remove the scattering. Some virus preps will
contain significant amounts of aggregated virus, which exacerbates the
scattering issue.
 
With respect to Carlos's point about empty capsids, it is possible to use
AUC to separate and quantitate empty from full capsids. The paper below by
Steve Berkowitz at Biogen-Idec and myself discusses that aspect as well as
the use of the interference optics to get the mass concentration, together
with a monomer mass from theoretical composition or AUC or whatever, to get
out the number concentration of total and/or filled capsids.
 
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.

 
John

  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Carlos E. Catalano
Sent: Thursday, March 04, 2010 7:48 AM
To: dandres at uni-potsdam.de
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] determination of phage particle concentration


sabine, 
we have used the EM method to quantify total number of particles using
polystyrene beads as an internal standard.  you must titer to quantify
infective particles, of course.  uv method will work fine, but assumes that
all of the absorbing species are actually proteins incorporated into a viral
particle (i.e., will also detect partially-assembled structures).
peter - the nanosight sounds interesting ...
cec

=========================================
Carlos Enrique Catalano, Pharm.D., Ph.D.
Professor of Medicinal Chemistry
University of Washington School of Pharmacy
172H Health Science Building
Box 357610
Seattle, WA  98195-7610
(206) 685-2468  (office)
(206) 685-3252  (fax)
catalanc at u.washington.edu
http://depts.washington.edu/medchem/faculty/Catalano.html






On Mar 4, 2010, at 2:57 AM, kaltofen at uni-potsdam.de wrote:

Dear all,

we are trying to obtain the concentration of bacteriophages in a solution,
concentration meaning the number of particles per ml. The solution is
supposed to be monodisperse and the MW is (roughly) known but could be
determined exactly. Is the any idea of how to do this with a hydrodynamic
method (staining is difficult to compare between phage mutants and counting
infected cells is tedious). I`ve got the "feeling" that one could use the
number-averaged MW, but maybe this is totally wrong.

Thank you for any hints, literature is also welcome.

Cheers,

Sabine




Sabine Kaltofen
PhD student

Universität Potsdam
Department of Physical Biochemistry
Institute of Biochemistry and Biology
Karl-Liebknecht-Str. 24-25, Haus 25, Raum B/0.05
D-14476 Potsdam-Golm
Telefon: +49-(331)-977-5245
Email: kaltofen at uni-potsdam.de



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