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<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Thanks John<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>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 size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>I am not familiar with competitor models
for direct boundary fitting.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Tom<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><b><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma;font-weight:bold'>From:</span></font></b><font
size=2 face=Tahoma><span style='font-size:10.0pt;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>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>Tom<o:p></o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'> <o:p></o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;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>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'> <o:p></o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'> <o:p></o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'> <o:p></o:p></span></font></p>
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<p class=MsoNormal style='margin:0cm;margin-bottom:.0001pt'><font size=2
face=Tahoma><span style='font-size:10.0pt;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>
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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>
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<p class=MsoNormal style='background:#F3F3F3'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Hello<o:p></o:p></span></font></p>
<p class=MsoNormal style='background:#F3F3F3'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='background:#F3F3F3'><font size=2 face=Arial><span
style='font-size:10.0pt;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 size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='background:#F3F3F3'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Thanks for any thoughts<o:p></o:p></span></font></p>
<p class=MsoNormal style='background:#F3F3F3'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='background:#F3F3F3'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Tom Jowitt<o:p></o:p></span></font></p>
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