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Samantha Jones (PRION) wrote:<br>
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<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">Hello<o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">I recently analyzed some
data using c(S) distribution model
on <span class="SpellE">Sedfit</span>. The distribution showed a few
peaks, with
the main peak being at 4S. I floated the frictional ratio and came back
with
approx 1.5. I then converted this to a <span class="GramE">c(</span>M)
distribution with the main peak now showing approx 65,000 </span></font><st1:City><st1:place><font
face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">Daltons</span></font></st1:place></st1:City><font
face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">. Knowing
our monomer weight as 16,400 </span></font><st1:City><st1:place><font
face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">Daltons</span></font></st1:place></st1:City><font
face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;"> I concluded
this was a tetramer.<o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">At the same time the same
protein sample was analyzed using synchrotron
<span class="GramE">X</span> ray scattering. Their results concluded
an <span class="SpellE">octomer</span>, a two ring structure.<o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">With this knowledge I
went back to the data and reanalyzed
using <span class="SpellE">Sedfit</span>, c(s) and <span class="GramE">c(</span>M)
again. Using the calculator function on <span class="SpellE">Sedfit</span>
I calculated
that for 4S with a frictional ratio 1.5 this would be roughly the
tetramer
weight, however, a frictional ratio of 2.4 for 4s gives a molecular
weight
equal to the <span class="SpellE">octomer</span>. When I entered this
into the
parameters of the <span class="GramE">c(</span>M) model the <span
class="SpellE">rsmd</span>
values show the f/f0 of 2.4 gives a better fit, 0.07 to 0.119 for f/f0
of 1.5.<o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">My question is how I
could have got this answer without the knowledge
of the scattering data? Is the use of the calculator function to fit
data
recommended? Especially in a sample with three peaks?<o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">Many thanks<o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;">Samantha <o:p></o:p></span></font></p>
<p class="MsoNormal"><font face="Arial" size="2"><span
style="font-size: 10pt; font-family: Arial;"><o:p> </o:p></span></font></p>
<p class="MsoAutoSig"><font color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB">Samantha Jones<o:p></o:p></span></font></p>
<p class="MsoAutoSig"><font color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB">MRC Prion Unit<o:p></o:p></span></font></p>
<p class="MsoAutoSig"><st1:place><st1:PlaceType><font color="purple"
face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB">Institute</span></font></st1:PlaceType><font
color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB"> of </span></font><st1:PlaceName><font color="purple"
face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB">Neurology</span></font></st1:PlaceName></st1:place><font
color="purple" face="Verdana" size="2"><span
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lang="EN-GB"><o:p></o:p></span></font></p>
<p class="MsoAutoSig"><font color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB">Queen Square<o:p></o:p></span></font></p>
<p class="MsoAutoSig"><st1:City><st1:place><font color="purple"
face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB">London</span></font></st1:place></st1:City><font
color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"
lang="EN-GB"><o:p></o:p></span></font></p>
<p class="MsoAutoSig"><font color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;">WC1N 3BG</span></font><font
color="purple" face="Verdana" size="2"><span
style="font-size: 11pt; font-family: Verdana; color: purple;"> <span
lang="EN-GB"><o:p></o:p></span></span></font></p>
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style="font-size: 12pt;"><o:p> </o:p></span></font></p>
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<font size="+1"><tt>Hello Samantha.<br>
My understanding of sedfit is that when you have a mixture of
molecular species in your AUC sample, the frictional ratio becomes very
hard to estimate for any one of the particular species. <br>
There are other techniques that might be better suited to evaluate the
molecular size of the individual species. You might consider two
dimensional gel electrophoresis using a gradient native gel in the
first dimension and an SDS gel in the second. The peak intensities and
the "molecular sizes" can be estimated to +/- 10% if you run standards
at the same time.<br>
all the best<br>
jack kornblatt<br>
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