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Hi Titus,<br>
<br>
It matters if, for example, you have a single channel of data that is
run at several different speeds and you tell your fitter that the
loading concentration is the same for all data sets (because it really
is). If you don't force that restriction, it doesn't matter but if the
Rm and Rb positions are not accurately specified, the effective loading
concentration will be different at each speed (i.e. each data set). If
you can specify that the loading concentration is the same for each
data set, then you can remove N-1 parameters, i.e. you fit for just one
loading concentration. So it helps if you can locate the bottom. But
John Philo has raised some important points, qv. Also if you have any
pelleting or gel fomation at the bottom, all bets are off and you will
have to float all loading concentrations anyway, even if you know the
exact positions of Rm and Rb.<br>
<br>
So as always - it depends ...<br>
<br>
Walter<br>
<br>
Titus M. Franzmann wrote:
<blockquote cite="mid:008001cb988e$da0cc8c0$8e265a40$@edu" type="cite">
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<p class="MsoNormal"><span style="color: rgb(31, 73, 125);">Hello
everyone, <o:p></o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);">I am
having trouble following this discussion. At what particular point do
these proper positions really become a matter? Is this a matter of “how
close to the bottom can I reliably analyze an Eq-dataset”? Would the
bottom position matter, if the absorbance close to the bottom is beyond
Abs=1, and only you analyze the radial range that covers the signal
below Abs=1?<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);">Best<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);">Titus<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);"><o:p> </o:p></span></p>
<div>
<div
style="border-style: solid none none; border-color: rgb(181, 196, 223) -moz-use-text-color -moz-use-text-color; border-width: 1pt medium medium; padding: 3pt 0in 0in;">
<p class="MsoNormal"><b><span
style="font-size: 10pt; font-family: "Tahoma","sans-serif"; color: windowtext;">From:</span></b><span
style="font-size: 10pt; font-family: "Tahoma","sans-serif"; color: windowtext;">
<a class="moz-txt-link-abbreviated" href="mailto:rasmb-bounces@server1.bbri.org">rasmb-bounces@server1.bbri.org</a> [<a class="moz-txt-link-freetext" href="mailto:rasmb-bounces@server1.bbri.org">mailto:rasmb-bounces@server1.bbri.org</a>] <b>On
Behalf Of </b>John Philo<br>
<b>Sent:</b> Friday, December 10, 2010 11:53 AM<br>
<b>To:</b> 'Walter Stafford'; 'Stephen Harding';
<a class="moz-txt-link-abbreviated" href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a><br>
<b>Subject:</b> Re: [RASMB] radial scans 6 sector cells<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;">Steve
& Walter, </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;">Well
I don't like to be belabor a point, but since I'm getting beat up a bit
here... </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;">Yes,
the conservation of mass calculations do need to know the meniscus and
bottom positions, but that doesn't mean those positions have to be
present in the final equilibrium scan you are actually fitting.
(Clearly they don't since I have been using this data
acquisition approach for many years, including when using my 'soft'
conservation of mass constraints). From an experimental point of view
it is also not very clear how to accurately define those locations from
scans that include them (e.g. the infamous "where is the meniscus,
really?" controversy).</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,
regarding the bottom position, what does the 'true' position actually
mean? The bottom of the standard Beckman 6-channel cells is flat, not
cut on a radius, so (without FC43) the length of the solution column
varies as you move across the width of the cell. Even when you use FC43
there is a radial region at the bottom where the absorbance optics are
detecting part sample solution and part FC43, so you really can't
possibly get good data for the entire solution column (this issue
should pretty much go away when you use interference slits). Often mass
conservation approaches are not actually considering the rectangular
rather than sector shape of the channel at all.</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;">These
issues of not knowing precisely where the column ends are located,
non-sector geometry, and not being able to get good data over the full
column height, are exactly why true accurate mass conservation usually
cannot be achieved and precisely why I implemented the 'soft' mass
conservation constraints about 16 years ago [Philo, J. S. (2000).
Improving sedimentation equilibrium analysis of mixed associations
using numerical constraints to impose mass or signal conservation. <i>Methods
Enzymol. </i>321, 100-120].</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;">John</span><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 class="moz-txt-link-abbreviated" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a class="moz-txt-link-freetext" href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On
Behalf Of </b>Walter Stafford<br>
<b>Sent:</b> Thursday, December 09, 2010 8:21 PM<br>
<b>To:</b> Stephen Harding; <a class="moz-txt-link-abbreviated" href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br>
<b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p>
<p class="MsoNormal">So I agree with Steve:<br>
<br>
Those data may not be recordable, especially near the bottom at higher
speeds, but one must know those positions accurately to do the curve
fitting properly (for two more components) because the best fit must
match the observable part of the data (i.e. that range over which the
data are being compared to the model for computation of the rms
residuals), and the proper best fit (i.e. the model) is computed
assuming those positions are known. For a self-associating system
(which is a single component, albeit multi-species) it is not so
important since the only error in that case would be the loading
concentration - which we're not especially interested in. But for a
hetero-associating system, say A+B=AB, the relative loading
concentrations of the two components do matter in the fitting and in
getting accurate values of the equilibrium constants. <br>
<br>
Since I do most of my runs using interference optics and always do a
blank run both before and after the main run in that case, at the speed
of the run, I can use those scans to get the positions of the bases of
the channels. And SEDANAL will do an interpolated blank subtraction.<br>
<br>
Walter<br>
<br>
Stephen Harding wrote: <o:p></o:p></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);">I also
disagree with John and agree almost 100% with Walter, i.e. meniscus and
base positions have to be identifiable (and not just for SEDANAL) but I
don’t think its possible to record (reliable) concentration data at the
solution boundaries, particularly for heterogeneous systems.</span><o:p></o:p></p>
<p class="MsoNormal"><span style="color: rgb(31, 73, 125);">Steve</span><o:p></o:p></p>
<div>
<div
style="border-style: solid none none; border-color: rgb(181, 196, 223) -moz-use-text-color -moz-use-text-color; border-width: 1pt medium medium; padding: 3pt 0in 0in;">
<p class="MsoNormal" style="margin-left: 0.5in;"><b><span
style="font-size: 10pt; font-family: "Tahoma","sans-serif"; color: windowtext;">From:</span></b><span
style="font-size: 10pt; font-family: "Tahoma","sans-serif"; color: windowtext;">
<a moz-do-not-send="true" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a>
[<a moz-do-not-send="true" href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>]
<b>On Behalf Of </b>Walter Stafford<br>
<b>Sent:</b> 09 December 2010 23:14<br>
<b>To:</b> <a moz-do-not-send="true" href="mailto:jphilo@mailway.com">jphilo@mailway.com</a><br>
<b>Cc:</b> <a moz-do-not-send="true"
href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br>
<b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p>
</div>
</div>
<p class="MsoNormal" style="margin-left: 0.5in;">I disagree with
John. <br>
<br>
For example, my software (SEDANAL) needs to have both the menicus and
the base positions identifiable in order to do consrevation of mass
calculations for multi-compnent systems. For an equilibrium run - since
time is not of the essentce here - is to collect enough data to include
the solution boundaries.<br>
<br>
actually, I usually collect all three channels of data in one scan and
process it afterward - once for each channel. Less muss and less fuss.<br>
<br>
<br>
<o:p></o:p></p>
<pre style="margin-left: 0.5in;"> <o:p></o:p></pre>
<pre style="margin-left: 0.5in;">===============================================<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">Walter F. Stafford, Ph.D.<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">Senior Scientist<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">Boston Biomedical Research Institute<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">64 Grove Street<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">Watertown, MA 02472<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">617-658-7808<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">skype: w.stafford3<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">----------------------<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">"Be kind, for everyone you meet is fighting a hard battle." -Plato<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">------------------------------------ <o:p></o:p></pre>
<blockquote style="margin-top: 5pt; margin-bottom: 5pt;">
<div style="margin-left: 0.5in;">
<div class="MsoNormal" style="text-align: center;" align="center"><span
style="font-size: 12pt; font-family: "Times New Roman","serif";">
<hr align="center" size="2" width="100%"></span></div>
</div>
<p class="MsoNormal"
style="margin-right: 0in; margin-bottom: 12pt; margin-left: 0.5in;"><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
moz-do-not-send="true" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a>
[<a moz-do-not-send="true" href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>]
<b>On Behalf Of </b>Lake Paul<br>
<b>Sent:</b> Thursday, December 09, 2010 1:26 PM<br>
<b>To:</b> 'Roy Hantgan'; <a moz-do-not-send="true"
href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br>
<b>Subject:</b> Re: [RASMB] radial scans 6 sector cells</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="color: rgb(31, 73, 125);">Roy,</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="color: rgb(31, 73, 125);">Funny you should mention this, I did
my first 6-sector experiment yesterday. The 5.7 default might not cover
the inside sector, so you might have to adjust it to 5.65. I did this
by trial and error and by getting help from Dr. John Burgner. The 5.65
worked well I get alittle of the top of the cell and both menisci. Hope
this helps.</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="color: rgb(31, 73, 125);">Lake</span><o:p></o:p></p>
<div>
<div
style="border-style: solid none none; border-color: windowtext -moz-use-text-color -moz-use-text-color; border-width: 1pt medium medium; padding: 3pt 0in 0in;">
<p class="MsoNormal" style="margin-left: 0.5in;"><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
moz-do-not-send="true" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a>
[<a moz-do-not-send="true" href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>]
<b>On Behalf Of </b>Roy Hantgan<br>
<b>Sent:</b> Thursday, December 09, 2010 3:42 PM<br>
<b>To:</b> <a moz-do-not-send="true"
href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a><br>
<b>Subject:</b> [RASMB] radial scans 6 sector cells</span><o:p></o:p></p>
</div>
</div>
<p class="MsoNormal" style="margin-left: 0.5in;">Dear colleagues:<o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;">Can you please
remind me how you set the radial distance for sed eq runs in 6-sector
cells?<br>
I’ve only run sed eq in double sector cells. <o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;">Thanks for your
advice,<o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;">Roy <o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="font-size: 10pt; font-family: "Arial","sans-serif";">Roy R.
Hantgan, Ph.D.</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="font-size: 10pt; font-family: "Arial","sans-serif";">Associate
Professor of Biochemistry / Associate in Molecular Medicine</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="font-size: 10pt; font-family: "Arial","sans-serif";">Director,
Macromolecular Interactions Core Laboratory (MICL)</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="font-size: 10pt; font-family: "Arial","sans-serif";">Wake
Forest University School of Medicine</span><o:p></o:p></p>
<p class="MsoNormal" style="margin-left: 0.5in;"><span
style="font-size: 10pt; font-family: "Arial","sans-serif";">3058 Hanes
Bldg / TEL 336-716-4675</span><o:p></o:p></p>
<pre style="margin-left: 0.5in;"> <o:p></o:p></pre>
<pre style="margin-left: 0.5in;"> <o:p></o:p></pre>
<pre style="margin-left: 0.5in;">_______________________________________________<o:p></o:p></pre>
<pre style="margin-left: 0.5in;">RASMB mailing list<o:p></o:p></pre>
<pre style="margin-left: 0.5in;"><a moz-do-not-send="true"
href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><o:p></o:p></pre>
<pre style="margin-left: 0.5in;"><a moz-do-not-send="true"
href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><o:p></o:p></pre>
<pre style="margin-left: 0.5in;"> <o:p></o:p></pre>
</blockquote>
<p class="MsoNormal"><span
style="font-size: 12pt; font-family: "Times New Roman","serif";"><o:p> </o:p></span></p>
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<o:p></o:p></span></p>
<pre> <o:p></o:p></pre>
<pre>-- <o:p></o:p></pre>
<pre><o:p> </o:p></pre>
<pre>===============================================<o:p></o:p></pre>
<pre>Walter F. Stafford, Ph.D.<o:p></o:p></pre>
<pre>Senior Scientist<o:p></o:p></pre>
<pre>Boston Biomedical Research Institute<o:p></o:p></pre>
<pre>64 Grove Street<o:p></o:p></pre>
<pre>Watertown, MA 02472<o:p></o:p></pre>
<pre>617-658-7808<o:p></o:p></pre>
<pre>skype: w.stafford3<o:p></o:p></pre>
<pre>----------------------<o:p></o:p></pre>
<pre>"Be kind, for everyone you meet is fighting a hard battle." -Plato<o:p></o:p></pre>
<pre>------------------------------------ <o:p></o:p></pre>
</div>
<pre wrap="">
<fieldset class="mimeAttachmentHeader"></fieldset>
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</pre>
</blockquote>
<br>
<pre class="moz-signature" cols="72">--
===============================================
Walter F. Stafford, Ph.D.
Senior Scientist
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472
617-658-7808
skype: w.stafford3
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"Be kind, for everyone you meet is fighting a hard battle." -Plato
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