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<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial>Sabine,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial>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.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial>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.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial>
<P style="MARGIN-TOP: 9px; MARGIN-BOTTOM: 0px">Berkowitz, S. A. and Philo, J. S.
(2007). Monitoring the homogeneity of adenovirus preparations (a gene therapy
delivery system) using analytical ultracentrifugation. <I>Anal. Biochem.</I>
362, 16-37.</P></FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=609413017-04032010><FONT color=#0000ff
size=2 face=Arial>John</FONT></SPAN></DIV><BR>
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<FONT size=2 face=Tahoma><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf Of </B>Carlos E.
Catalano<BR><B>Sent:</B> Thursday, March 04, 2010 7:48 AM<BR><B>To:</B>
dandres@uni-potsdam.de<BR><B>Cc:</B> rasmb@server1.bbri.org<BR><B>Subject:</B>
Re: [RASMB] determination of phage particle concentration<BR></FONT><BR></DIV>
<DIV></DIV>sabine,
<DIV>we have used the EM method to quantify total number of particles using
polystyrene beads as an internal standard. you must titer to quantify
<I>infective</I> 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).</DIV>
<DIV>peter - the nanosight sounds interesting ...</DIV>
<DIV>cec<BR>
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<DIV>=========================================</DIV>
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Enrique Catalano, Pharm.D., Ph.D.</SPAN></SPAN></FONT></DIV>
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<DIV>On Mar 4, 2010, at 2:57 AM, <A
href="mailto:kaltofen@uni-potsdam.de">kaltofen@uni-potsdam.de</A>
wrote:</DIV><BR class=Apple-interchange-newline>
<DIV>Dear all,<BR><BR>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.<BR><BR>Thank you for
any hints, literature is also
welcome.<BR><BR>Cheers,<BR><BR>Sabine<BR><BR><BR><BR><BR>Sabine Kaltofen<BR>PhD
student<BR><BR>Universität Potsdam<BR>Department of Physical
Biochemistry<BR>Institute of Biochemistry and Biology<BR>Karl-Liebknecht-Str.
24-25, Haus 25, Raum B/0.05<BR>D-14476 Potsdam-Golm<BR>Telefon:
+49-(331)-977-5245<BR>Email: <A
href="mailto:kaltofen@uni-potsdam.de">kaltofen@uni-potsdam.de</A><BR><BR><BR><BR>_______________________________________________<BR>RASMB
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