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<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2>Tom,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2>I agree with Jack Correia's points but I am not sure he
stated forcefully enough that the <U>heights</U> of the peaks are
irrelevant---it is only the areas that are potentially meaningful. The peak
widths from the c(s) method depend on signal/noise ratio, maximum entropy
settings, etc. and in general have no direct physical meaning, and therefore the
same is true for the peak heights. </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2>I'm also not sure you and Jack are using the
term "normalization" in the same sense. The normalization Jack is referring
to sets the total area under the curve of a g(s*) or c(s) distribution to 1
(100%) so the area for each peak gives the fraction of that species. You can
manually normalize a c(s) distribution from SEDFIT by exporting it and dividing
the Y values by the total area reported by SEDFIT's integration function; in
DCDT+ normalization is a built-in option.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2>With regard to fitting SV data to an explicit competition
model, since SEDANAL allows developing and fitting your own assembly models I am
sure it could do this.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=482000916-20052008><FONT face=Arial
color=#0000ff size=2>John</FONT></SPAN></DIV><BR>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left>
<HR tabIndex=-1>
<FONT face=Tahoma size=2><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf Of </B>Thomas
Jowitt<BR><B>Sent:</B> Tuesday, May 20, 2008 8:55 AM<BR><B>To:</B> John Correia;
rasmb@rasmb.bbri.org<BR><B>Subject:</B> RE: [RASMB] Sedfit peak
integration<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV class=Section1>
<P class=MsoNormal><FONT face=Arial color=navy size=2><SPAN
style="FONT-SIZE: 10pt; COLOR: navy; FONT-FAMILY: Arial">Thanks
John<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal><FONT face=Arial color=navy size=2><SPAN
style="FONT-SIZE: 10pt; COLOR: navy; FONT-FAMILY: Arial">This is a slowly
interacting system that we have characterized using the kds from sed eq. however
it is complicated by the fact that it is partially calcium dependant, although
they do interact slowly when no calcium is present (at least non-added). I will
have a look at the DCDT plots and try to normalize them there. We are attempting
to compete with fragments of the same molecule to establish which domains are
responsible for dimerisation, therefore no color unfortunately. You are right in
that the concentration of competition fragments is critical and we can see
competition with some of the fragments. Only quantifying between different runs
at different wavelengths is tricky. <o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal><FONT face=Arial color=navy size=2><SPAN
style="FONT-SIZE: 10pt; COLOR: navy; FONT-FAMILY: Arial">I am not familiar with
competitor models for direct boundary fitting.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal><FONT face=Arial color=navy size=2><SPAN
style="FONT-SIZE: 10pt; COLOR: navy; FONT-FAMILY: Arial">Tom<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal><FONT face=Arial color=navy size=2><SPAN
style="FONT-SIZE: 10pt; COLOR: navy; FONT-FAMILY: Arial"><o:p> </o:p></SPAN></FONT></P>
<DIV>
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<HR tabIndex=-1 align=center width="100%" SIZE=2>
</SPAN></FONT></DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><B><FONT face=Tahoma size=2><SPAN
style="FONT-WEIGHT: bold; FONT-SIZE: 10pt; FONT-FAMILY: Tahoma">From:</SPAN></FONT></B><FONT
face=Tahoma size=2><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma"> John
Correia [mailto:jcorreia@biochem.umsmed.edu] <BR><B><SPAN
style="FONT-WEIGHT: bold">Sent:</SPAN></B> 20 May 2008 15:34<BR><B><SPAN
style="FONT-WEIGHT: bold">To:</SPAN></B> thomas.a.jowitt@manchester.ac.uk;
rasmb@rasmb.bbri.org<BR><B><SPAN style="FONT-WEIGHT: bold">Subject:</SPAN></B>
Re: [RASMB] Sedfit peak integration</SPAN></FONT><o:p></o:p></P></DIV>
<P class=MsoNormal><FONT face="Times New Roman" size=3><SPAN
style="FONT-SIZE: 12pt"><o:p> </o:p></SPAN></FONT></P>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma">Tom<o:p></o:p></SPAN></FONT></P></DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma"> <o:p></o:p></SPAN></FONT></P></DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma">You described a noninteracting or
slowly interacting system. A reversible monomer dimer system is one peak,
skewed near the mid point of the transition. For a reversible system plot
Sw vs total concentrations to observe the extent of dimerization. This
requires a Kd in the accessible concentration regime. If you see two
peaks its nonreversible or slow kinetics, possibly disulfide crosslinking.
Tight competitive binding with an inhibitor might produce a free monomer zone
but the expected shape or resolution into two zones will depend upon the
relative affinity of competitor with Kd for dimerization. A simple
assumption in your analysis may be too simple. Direct
boundary fitting to a competitive model is also possible. Does the
inhibitor have color? Knowing inhibitor concentration is a critical
feature for this analysis. Normalization of the peaks often makes it
easier to see the transition or changes in boundary shape but this is a
qualitative analysis. Use the total area under the curves to
normalize. Some programs like DCDT+ have a normalization function built
in.<o:p></o:p></SPAN></FONT></P></DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma"> <o:p></o:p></SPAN></FONT></P></DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma"> <o:p></o:p></SPAN></FONT></P></DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma"> <o:p></o:p></SPAN></FONT></P></DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><FONT face=Tahoma size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma">-------------------------------------------------------------------<BR>Dr.
John J. "Jack" Correia<BR>Department of Biochemistry<BR>University of <st1:place
w:st="on"><st1:PlaceName w:st="on">Mississippi</st1:PlaceName> <st1:PlaceName
w:st="on">Medical</st1:PlaceName> <st1:PlaceType
w:st="on">Center</st1:PlaceType></st1:place><BR><st1:Street
w:st="on"><st1:address w:st="on">2500 North State
Street</st1:address></st1:Street><BR><st1:place w:st="on"><st1:City
w:st="on">Jackson</st1:City>, <st1:State w:st="on">MS</st1:State>
<st1:PostalCode w:st="on">39216</st1:PostalCode></st1:place><BR>(601)
984-1522
<BR>fax (601)
984-1501
<BR>email address: jcorreia@biochem.umsmed.edu
<BR>homepage location: <A
href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</A><BR>dept
homepage location: <A
href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</A><BR>-------------------------------------------------------------------<BR><BR><BR><BR><BR>>>>
On 5/20/2008 at 9:05 AM, in message
<20080520150555687.00000003664@ThomasJowitt>, "Thomas Jowitt"
<thomas.a.jowitt@manchester.ac.uk>
wrote:<o:p></o:p></SPAN></FONT></P></DIV>
<DIV
style="BORDER-RIGHT: medium none; PADDING-RIGHT: 0cm; BORDER-TOP: medium none; PADDING-LEFT: 5pt; PADDING-BOTTOM: 0cm; MARGIN-LEFT: 11.25pt; BORDER-LEFT: #050505 1pt solid; PADDING-TOP: 0cm; BORDER-BOTTOM: medium none">
<DIV>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial">Hello<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial"><o:p> </o:p></SPAN></FONT></P>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial">I have a question regarding peak
integration between different sedfit analyses. In an effort to gauge competition
in a self-associating system, velocity was used to assess the level of
dimerisation and subsequent competition using different concentrations of dimer
and the competing element. The question is one of assessing the relative degree
of competition, therefore the integration or relative size of the peaks. Is it
right to normalize the different runs to the height of the dimer (predominant
species), and therefore compare the relative size of the monomer peaks for
competition, and if so can the integrated value of the normalized peaks be used
also, or is that more subjective?<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial"><o:p> </o:p></SPAN></FONT></P>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial">Thanks for any
thoughts<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial"><o:p> </o:p></SPAN></FONT></P>
<P class=MsoNormal style="BACKGROUND: #f3f3f3"><FONT face=Arial size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: Arial">Tom
Jowitt<o:p></o:p></SPAN></FONT></P></DIV></DIV>
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