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<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial>John S., first I think all responders have been assuming that
in your SEDANAL single-species fit you were fitting the entire run (all the
scans where something is still in the cell), and then also doing the same in
your <EM>c(s)</EM> fitting. If that is not correct then the picture is
different. And obviously you shouldn't directly compare a single species fit
from SEDFIT to the one from SEDANAL unless you are fitting the same scans. But I
think you must have been fitting the entire run in SEDANAL or you would not have
gotten such a gross over-estimate of <EM>D</EM> and under-estimate of
<EM>M.</EM></FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial>Yes if you really have 20% of other species in the sample then
I would not expect that you would get a good fit of the entire run as a single
species, yet I think that is what you said about the SEDANAL results. Anyway no
I was not proposing that you would try to select a subset of scans to focus only
on the main boundary. Yes you could effectively cut out the faster-sedimenting
minor species by eliminating the early scans from the fit, but any
slowly-sedimenting minor components will always be in the cell whenever the main
boundary still is.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial>The <EM>g(s*)</EM> procedure I was proposing to effectively
ignore the minor components and focus the analysis on the major species is not
something you can do in SEDANAL---you can only do this in DCDT+, or
by porting the <EM>g(s*)</EM> distribution into some other software and
fitting with an appropriate function. The basic idea is that after transforming
the raw data into sedimentation coefficient space you can reduce the effects of
the slow- or fast-sedimenting junk in the sample by simply chopping
off the upper and lower ends of the <EM>g(s*)</EM> distribution and fitting only
the middle part of the main peak (to a single species model). An example of this
approach is shown in Fig. 6 of reference 1 below, and then ref 2 has a more
extensive discussion about it and a validation of the statistical estimates of
its precision via 15 replicates of one experiment.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial>Also FYI the fact that fitting a heterogeneous sample as a
single species in whole boundary analysis leads to significant errors in
estimating mass and diffusion coefficient was discussed in the first paper
on whole boundary analysis with personal computers, ref. 3 from 1994. When
a sample of BSA containing ~10% dimer was fitted as a single species the
monomer mass was low by 20%.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial>John P.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
size=2 face=Arial>------------</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT color=#0000ff
face=Arial><FONT size=2><FONT color=#000000>(1)</FONT> <FONT
color=#000000>Philo, J. S. (2006). Improved methods for fitting sedimentation
coefficient distributions derived by time-derivative techniques. Anal. Biochem.
354, 238-246 <A
href="http://www.jphilo.mailway.com/Anal_Biochem_gofs_fitting_2006.pdf">http://www.jphilo.mailway.com/Anal_Biochem_gofs_fitting_2006.pdf</A></FONT></FONT></FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT size=2
face=Arial>(2) Philo, J. S. (2011). Limiting the sedimentation coefficient for
sedimentation velocity data analysis: Partial boundary modeling and <I>g(s*)</I>
approaches revisited. <I>Anal. Biochem.</I> 412, 189-202 </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=426273720-13082013><FONT size=2
face=Arial>(3) <FONT size=2>Philo, J. S. (1994). Measuring sedimentation,
diffusion, and molecular weights of small molecules by direct fitting of
sedimentation velocity concentration profiles. In: <I>Modern analytical
ultracentrifugation</I>. T.M.Schuster and T.M.Laue, eds. Birkhauser, Boston, pp.
156-170 <A
href="http://www.jphilo.mailway.com/svedberg.pdf">http://www.jphilo.mailway.com/svedberg.pdf</A></FONT></FONT></SPAN></DIV>
<DIV dir=ltr align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> John Sumida
[mailto:jpsumida@u.washington.edu] <BR><B>Sent:</B> Tuesday, August 13, 2013
12:25 PM<BR><B>To:</B> jphilo@mailway.com; rasmb@rasmb.org<BR><B>Cc:</B> 'Walter
Stafford'<BR><B>Subject:</B> RE: [RASMB] MW calculation
question<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV class=WordSection1>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">Dear John P.
Your comment regarding the effects of fitting the whole set of boundary data is
very helpful. <o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 14pt; COLOR: blue"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">But just to make
sure I am clear on this, I believe what you are saying is to use dcdt in order
to determine the dataset that is consistent with the major (80%) species
observed in sedfit, and then proceed with the single species model in SEDANAL?
<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 14pt; COLOR: blue"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">I will also check
the single species fit in SEDFIT – I had not tried that yet – but I think that
this also should only use those scans that correspond to the single species
otherwise the fit could prove to be quite poor?<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 14pt; COLOR: blue"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">Thank you all for
all the comments. They are not only helpful and informative but
encouraging as well.<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 14pt; COLOR: blue"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">Best
regards<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">John
Sumida<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">Analytical
Biopharmacy Core<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 14pt; COLOR: blue">University of
Washington.<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 14pt; COLOR: blue"><o:p> </o:p></SPAN></P>
<DIV>
<DIV
style="BORDER-TOP: #b5c4df 1pt solid; BORDER-RIGHT: medium none; BORDER-BOTTOM: medium none; PADDING-BOTTOM: 0in; PADDING-TOP: 3pt; PADDING-LEFT: 0in; BORDER-LEFT: medium none; PADDING-RIGHT: 0in">
<P class=MsoNormal><B><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Tahoma','sans-serif'">From:</SPAN></B><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Tahoma','sans-serif'"> John Philo
[mailto:jphilo@mailway.com] <BR><B>Sent:</B> Tuesday, August 13, 2013 10:05
AM<BR><B>To:</B> rasmb@rasmb.org<BR><B>Cc:</B> 'Walter Stafford'; 'John
Sumida'<BR><B>Subject:</B> RE: [RASMB] MW calculation
question<o:p></o:p></SPAN></P></DIV></DIV>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'; COLOR: blue">John S.,
I agree with Walter Stafford that heterogeneity is causing the single-species
fit to underestimate the mass, but I don't think there is any reason to think
the primary cause is heterogeneity of monomer mass or conformation. You
said yourself that the <EM><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'">c(s)</SPAN></EM> analysis gives one
major peak that is only 80% of the total area. If the sample is only 80% pure,
why would you expect a single-species fit to give the correct MW?
If <EM><SPAN style="FONT-FAMILY: 'Arial','sans-serif'">c(s)</SPAN></EM> is
giving one narrow main peak then it is not its ability to emulate multiple
overlapping species that is primarily improving the fit quality relative to
a single-species fit, it is the addition of the 20% of minor
species (presumably with significantly different sedimentation
coefficients). </SPAN><o:p></o:p></P>
<P class=MsoNormal> <o:p></o:p></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'; COLOR: blue">The
strength of whole-boundary analysis is that it fits all the scans, but that
means that to get good results it must account for all species that are present,
whether or not they are things you are interested in! That is, a weakness of
whole boundary analysis is that it isn't particularly easy to exclude
impurities, aggregates, fragments or other 'junk' from the data analysis, and
your results simply demonstrate that problem. This is a case where a quick and
simple <EM><SPAN style="FONT-FAMILY: 'Arial','sans-serif'">g(s*)
</SPAN></EM>analysis via the dc/dt approach, and then fitting only the main peak
of that distribution (excluding the junk at high and low sedimentation
coefficients) would probably correctly identify the MW of your 80% major species
(but always the best precision should come from whole boundary
analysis).</SPAN><o:p></o:p></P>
<P class=MsoNormal> <o:p></o:p></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'; COLOR: blue">Further,
fundamentally your question is an apples vs. oranges comparison. You say you
want to compare the two different analysis programs, but you are trying to
compare two fundamentally different fitting models. You can easily do a
single-species fit in SEDFIT and then compare that to the one from SEDANAL (and
I'm sure the results will be similar).</SPAN><o:p></o:p></P>
<DIV>
<P class=MsoNormal> <o:p></o:p></P></DIV>
<DIV>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'; COLOR: blue">John
P.</SPAN><o:p></o:p></P></DIV>
<P class=MsoNormal><o:p> </o:p></P>
<DIV class=MsoNormal style="TEXT-ALIGN: center" align=center>
<HR align=center SIZE=2 width="100%">
</DIV>
<P class=MsoNormal style="MARGIN-BOTTOM: 12pt"><B><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Tahoma','sans-serif'">From:</SPAN></B><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Tahoma','sans-serif'"> <A
href="mailto:rasmb-bounces@list.rasmb.org">rasmb-bounces@list.rasmb.org</A> [<A
href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</A>]
<B>On Behalf Of </B>Walter Stafford<BR><B>Sent:</B> Monday, August 12, 2013 6:30
PM<BR><B>To:</B> John Sumida<BR><B>Cc:</B> <<A
href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</A>><BR><B>Subject:</B> Re:
[RASMB] MW calculaiton question</SPAN><o:p></o:p></P>
<DIV>
<P class=MsoNormal>I think this was already answered. I think your sample is
heterogeneous. Small variations on composition (I.e molar mass heterogeneity)
will cause peak broadening that will appear as diffusion. If you include enough
data in the fit. It should be apparent in the residuals. C(s) will add enough
components to account for the heterogeneity (albeit with the same f/fo) and
should give better residuals. Sedanal will not because fitting to a single
species (I. E, the wrong model) should not give as good of a fit. The
heterogeneity will cause Sedanal to return a value that is too large and
therefore, a molar mass that is too small. <BR><BR>Walter Stafford
<o:p></o:p></P>
<DIV>
<P class=MsoNormal><A
href="mailto:wstafford3@walterstafford.com">wstafford3@walterstafford.com</A><o:p></o:p></P></DIV>
<DIV>
<P class=MsoNormal><o:p> </o:p></P></DIV></DIV>
<DIV>
<P class=MsoNormal style="MARGIN-BOTTOM: 12pt"><BR>On Aug 12, 2013, at 20:57,
"John Sumida" <<A
href="mailto:jpsumida@u.washington.edu">jpsumida@u.washington.edu</A>>
wrote:<o:p></o:p></P></DIV>
<BLOCKQUOTE style="MARGIN-BOTTOM: 5pt; MARGIN-TOP: 5pt">
<DIV>
<P class=MsoPlainText>Dear RASMB,<o:p></o:p></P>
<P class=MsoPlainText>The following was posted on the SEDFIT user list and it
has been suggested that I also post this message to the RASMB list. Thus
I am reposting my question to RASMB and am including some edits and additional
information for clarity and correctness.<o:p></o:p></P>
<P class=MsoPlainText>Objective<o:p></o:p></P>
<P class=MsoPlainText>I am trying to reconcile results obtained using two
different approaches, SEDANAL and SEDFIT.<o:p></o:p></P>
<P class=MsoPlainText>Observations<o:p></o:p></P>
<P class=MsoPlainText>In SEDFIT, analysis of sed velocity FDS data returns an
s20w of 8.5s and MW=1.3 MDa for the major peak, comprising >80% of the
total loading concentration, determined in a c(s) fit. Please note after
checking with our collaborators this value is consistent with the MW returned
from DLS and static light scattering experiments. Thus the SEDFIT result
is consistent with the DLS and static light scattering data which estimate a
MW of 1.1 MDa. <o:p></o:p></P>
<P class=MsoPlainText>In SEDANAL, the simplest model necessary to fit the data
well was a single species model. Using this model, analysis of the same
data-set returns an s20w of 8.8s, similar to the value calculated in SEDFIT,
but a MW of 655 kDa is calculated.<o:p></o:p></P>
<P class=MsoPlainText>My question:<o:p></o:p></P>
<P class=MsoPlainText>Why does the value of the MW returned for SEDANAL and
SEDFIT differ by a factor of ~2 when the s20w in each case are similar (8.5s
versus 8.8s). My purpose here is that I believe the apparent difference
being returned from these parallel analyses is saying something fundamentally
important about the behavior of these materials in buffer and our assumptions
thereof.<o:p></o:p></P>
<P class=MsoPlainText>Background.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Rotor speed was 30 krpm, vbar=0.917
(measured), solution density = 1.00506 g/cm3 (measured), and viscosity
=0.0100281 Poise (measured). Temperature = 20oC.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
material being studies is a polymer micelle with a CMC of 14 micrograms/ml.
<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">3.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
polymer was run over a series of concentrations ranging from 0.2 mgs/ml to
1.00 mgs/ml. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Thus under the conditions of the
experiment I am at least 14 times the CMC at the lowest
concentration.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">4.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->A
major peak is observed in the initial c(s) distribution comprising >80% of
the total loading and the position of this peak (7.7 s-exp; s20w=8.5) does not
shift with concentration. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">5.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->From the analysis of raw SV data,
SEDANAL returns an experimental sedimentation coefficient of 7.9s and a
calculated s20w of 8.8s. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">6.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->A
global analysis in SEDPHAT was performed over the entire concentration range,
transforming the initial c(s) distribution into a set of 8 discrete
species. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Four of these 8 species survived a
critical chi square analysis suggesting that these four species were important
in retaining the quality of the fit. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->Of
these four discrete species, three were grouped beneath the major peak
observed in the initial c(s) analysis.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">3.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
calculated weight averaged s20w for these three species was 8.1s.
<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">4.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->The s20w values were checked and
confirmed with a manual calculation, SEDNTERP verstion 1.09, as well as the
calculate “s(20,w) from s(xp) in SEDFIT.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">7.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Using the ratio of s/D in the Svedberg
equation and values for diffusion estimated by assuming 655 kDA, (the MW
returned in SEDANAL), I calculate a MW of 655 kDa. Thus I believe that
the value for diffusion being estimated in SEDANAL is consistent with the 655
kDA molecular weight.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Estimates of the diffusion coefficient
in SEDFIT and SEDNTERP (and thus presumable also from SEDANAL) are not very
different. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
diffusion coefficient estimate in SEDFIT is 3.19E-7 cm2/sec and the value
calculated in SEDNTERP 3.67E-7 cm2/sec (Teller approximation).<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">8.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Using the calculate M(s) function in
SEDFIT and providing the experimental sedimentation coefficient noted above I
need to input an ff0<1 (0.8967) in order to arrive at the 655kDA MW
returned by SEDANAL. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->I
understand that this is nonsensical as ff0 cannot be less than 1.
<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Notably SEDNTERP version 1.09 also
calculates a frictional ratio<1; namely SEDNTERP calculate ffp=0.9449.
<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo2"><![if !supportLists]><SPAN
style="mso-list: Ignore">3.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
c(s) analysis in SEDFIT returns an ff0=1.47.<o:p></o:p></P>
<P class=MsoPlainText>Thus to summarize:<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->Relatively similar s20w values are
determined in both SEDFIT and SEDANAL for the same data-set.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->8.5s from SEDFIT<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->8.8s from SEDANAL <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->SEDFIT calculates a MW almost exactly
2 times the value of the MW returned by SEDANAL for the same
s20w.<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">3.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
estimates of diffusion in both programs are quite similar<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">4.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
frictional ratio ff0 in the SEDFIT analysis is 1.47 whereas the calculated ffp
in SEDNTERP based on the observe MW in SEDANAL is <1. <o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">5.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if !supportLists]--><!--[endif]-->The
frictional ratio calculated in SEDNTERP, assuming the MW=1.3 MDa, is
1.54<o:p></o:p></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo4"><![if !supportLists]><SPAN
style="mso-list: Ignore">6.<SPAN
style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN><![endif]><!--[if
!supportLists]--><!--[endif]-->There does appear to be heterogeneity
in the major peak observed in the c(s) analysis.<o:p></o:p></P>
<P class=MsoPlainText>Thank you in advance for your comments and
suggestions. I apologize for the length of this post, but in fairness, I
am attempting to provide information that would enable an informed
response.<o:p></o:p></P>
<P class=MsoPlainText>Best regards<o:p></o:p></P>
<P class=MsoPlainText>John Sumida<o:p></o:p></P>
<P class=MsoPlainText>University of Washington<o:p></o:p></P>
<P class=MsoPlainText>Analytical Biopharmacy
Core<o:p></o:p></P></DIV></BLOCKQUOTE>
<BLOCKQUOTE style="MARGIN-BOTTOM: 5pt; MARGIN-TOP: 5pt">
<DIV>
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