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<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial>Steve & Walter, </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial>Well I don't like to be belabor a point, but since I'm
getting beat up a bit here... </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial>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).</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial>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.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN><SPAN class=042271416-10122010><FONT
color=#0000ff size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial>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 [<FONT size=2>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</FONT>].</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=042271416-10122010><FONT color=#0000ff
size=2 face=Arial>John</FONT></SPAN></DIV>
<DIV dir=ltr align=left>
<HR tabIndex=-1>
</DIV>
<DIV dir=ltr align=left><FONT size=2 face=Tahoma><B>From:</B>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf
Of </B>Walter Stafford<BR><B>Sent:</B> Thursday, December 09, 2010 8:21
PM<BR><B>To:</B> Stephen Harding; rasmb@rasmb.bbri.org<BR><B>Subject:</B> Re:
[RASMB] radial scans 6 sector cells<BR></FONT><BR></DIV>
<DIV></DIV>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:
<BLOCKQUOTE
cite=mid:44005B520511CA40807C5D097783A29123393B4A63@EXCHANGE2.ad.nottingham.ac.uk
type="cite">
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<DIV class=WordSection1>
<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.<O:P></O:P></SPAN></P>
<P class=MsoNormal><SPAN
style="COLOR: rgb(31,73,125)">Steve<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-BOTTOM: medium none; BORDER-LEFT: medium none; PADDING-BOTTOM: 0cm; PADDING-LEFT: 0cm; PADDING-RIGHT: 0cm; BORDER-TOP: rgb(181,196,223) 1pt solid; BORDER-RIGHT: medium none; PADDING-TOP: 3pt">
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; COLOR: windowtext; FONT-SIZE: 10pt"
lang=EN-US>From:</SPAN></B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; COLOR: windowtext; FONT-SIZE: 10pt"
lang=EN-US> <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> 09 December 2010
23:14<BR><B>To:</B> <A class=moz-txt-link-abbreviated
href="mailto:jphilo@mailway.com">jphilo@mailway.com</A><BR><B>Cc:</B> <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<O:P></O:P></SPAN></P></DIV></DIV>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal>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><O:P></O:P></P><PRE style="MARGIN-LEFT: 36pt"><O:P> </O:P></PRE><PRE style="MARGIN-LEFT: 36pt">===============================================<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">Walter F. Stafford, Ph.D.<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">Senior Scientist<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">Boston Biomedical Research Institute<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">64 Grove Street<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">Watertown, MA 02472<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">617-658-7808<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">skype: w.stafford3<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">----------------------<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">"Be kind, for everyone you meet is fighting a hard battle." -Plato<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">------------------------------------ <O:P></O:P></PRE>
<BLOCKQUOTE style="MARGIN-TOP: 5pt; MARGIN-BOTTOM: 5pt">
<DIV style="TEXT-ALIGN: center; MARGIN-LEFT: 36pt" class=MsoNormal
align=center><SPAN
style="FONT-FAMILY: 'Times New Roman','serif'; FONT-SIZE: 12pt" lang=EN-US>
<HR align=center SIZE=2 width="100%">
</SPAN></DIV>
<P style="MARGIN-BOTTOM: 12pt; MARGIN-LEFT: 36pt; MARGIN-RIGHT: 0cm"
class=MsoNormal><B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt"
lang=EN-US>From:</SPAN></B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt" lang=EN-US> <A
href="mailto:rasmb-bounces@rasmb.bbri.org"
moz-do-not-send="true">rasmb-bounces@rasmb.bbri.org</A> [<A
href="mailto:rasmb-bounces@rasmb.bbri.org"
moz-do-not-send="true">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 href="mailto:rasmb@rasmb.bbri.org"
moz-do-not-send="true">rasmb@rasmb.bbri.org</A><BR><B>Subject:</B> Re:
[RASMB] radial scans 6 sector cells</SPAN><SPAN
style="FONT-FAMILY: 'Times New Roman','serif'; FONT-SIZE: 12pt"
lang=EN-US><O:P></O:P></SPAN></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="COLOR: rgb(31,73,125)">Roy,</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><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 style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="COLOR: rgb(31,73,125)">Lake</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="COLOR: rgb(31,73,125)"></SPAN><O:P></O:P></P>
<DIV>
<DIV
style="BORDER-BOTTOM: medium none; BORDER-LEFT: medium none; PADDING-BOTTOM: 0cm; PADDING-LEFT: 0cm; PADDING-RIGHT: 0cm; BORDER-TOP: 1pt solid; BORDER-RIGHT: medium none; PADDING-TOP: 3pt">
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt">From:</SPAN></B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt"> <A
href="mailto:rasmb-bounces@rasmb.bbri.org"
moz-do-not-send="true">rasmb-bounces@rasmb.bbri.org</A> [<A
href="mailto:rasmb-bounces@rasmb.bbri.org"
moz-do-not-send="true">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 href="mailto:rasmb@rasmb.bbri.org"
moz-do-not-send="true">rasmb@rasmb.bbri.org</A><BR><B>Subject:</B> [RASMB]
radial scans 6 sector cells</SPAN><O:P></O:P></P></DIV></DIV>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal>Dear colleagues:<O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal>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 style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal>Thanks for your
advice,<O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal>Roy <O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Roy R.
Hantgan, Ph.D.</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Associate
Professor of Biochemistry / Associate in Molecular
Medicine</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Director,
Macromolecular Interactions Core Laboratory (MICL)</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Wake Forest
University School of Medicine</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">3058 Hanes Bldg /
TEL 336-716-4675</SPAN><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P>
<P style="MARGIN-LEFT: 36pt" class=MsoNormal><O:P></O:P></P><PRE style="MARGIN-LEFT: 36pt"><O:P> </O:P></PRE><PRE style="MARGIN-LEFT: 36pt"><O:P> </O:P></PRE><PRE style="MARGIN-LEFT: 36pt">_______________________________________________<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt">RASMB mailing list<O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt"><A href="mailto:RASMB@rasmb.bbri.org" moz-do-not-send="true">RASMB@rasmb.bbri.org</A><O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt"><A href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb" moz-do-not-send="true">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</A><O:P></O:P></PRE><PRE style="MARGIN-LEFT: 36pt"> <O:P></O:P></PRE></BLOCKQUOTE></DIV><BR>
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--
===============================================
Walter F. Stafford, Ph.D.
Senior Scientist
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472
617-658-7808
skype: w.stafford3
----------------------
"Be kind, for everyone you meet is fighting a hard battle." -Plato
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