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<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2>Tara,</FONT></SPAN></DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff size=2>You
are making the graphs correctly, but your own statements about your results
show why these lines have different slopes. When you use the sigma calculated by
SEDNTERP using the sequence mass for a trimer, then your graph will give a line
corresponding to that sequence mass. However, you said that the fit from NONLIN
corresponds to a molecular mass that is 4.8% different from the sequence mass
for trimer, and one can easily see a 4.8% difference in slope on such a
graph. So yes, of course, the line using the NONLIN result goes through the data
very well (by definition if it is a good fit), and the line using the sequence
mass won't go through the data as well because your data don't
actually correspond to the molecular mass for pure trimer.</FONT></SPAN></DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff size=2>If you
are trying to show the expected slopes that correspond to trimer, dimer, and
tetramer then you should use the values based on the known monomer mass. But
fundamentally the graph isn't coming out like the published results because your
data is telling you the sample is not pure trimer (unless the vbar or density
are way off, but presumably you are using the same vbar as the other labs
used).</FONT></SPAN></DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff size=2>You
didn't say whether the NONLIN result is above or below the
expected trimer mass. If it is below, it might simply be because at the
concentration you used there is significant reversible dissociation to monomer
(perhaps the other labs were plotting data taken at a higher concentration,
where the fraction monomer is very low). If the concentrations are similar, and
your mass is low, I'm going to guess your sample is a mixture of trimer and some
denatured 'incompetent monomer' that cannot associate to form trimers. On the
other hand, if NONLIN says the average mass is above that for trimer, your
sample probably contains some aggregate.</FONT></SPAN></DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff size=2>'Hope
this helps,</FONT></SPAN></DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff size=2>John
Philo</FONT></SPAN></DIV>
<DIV><SPAN class=443044822-13102004><FONT face=Arial color=#0000ff
size=2>Alliance Protein Laboratories</FONT></SPAN></DIV>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">
<DIV></DIV>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left><FONT
face=Tahoma size=2>-----Original Message-----<BR><B>From:</B>
rasmb-admin@server1.bbri.org [mailto:rasmb-admin@server1.bbri.org] <B>On
Behalf Of </B>Tara Suntoke<BR><B>Sent:</B> Wednesday, October 13, 2004 10:48
AM<BR><B>To:</B> rasmb@server1.bbri.org<BR><B>Subject:</B> [RASMB] graphing
AUC data<BR><BR></FONT></DIV>
<DIV>Hi all,</DIV>
<DIV>I am fairly new to AUC and have a question about Sednterp and graphing
AUC data. </DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV>I spun a peptide that is a clean trimer- other people have published the
same result for the same peptide. I think the data is good for the
following reasons: the residuals are random, the data fits best to a
single species, and has a square root of variance of 5.9 e-3. In
addition, the molecular weight I get from the experiment, (24.8kD for the
trimer) is within 4.8% of the calculated MW in Sednterp (based on
composition). </DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV>I am trying to make a graph of ln(A) vs. r^2, and show that the peptide
corresponds to a trimer and not a dimer or tetramer. In order
to graph the lines of the dimer, trimer, and tetramer, I used the
equation of a line as follows:</DIV>
<DIV> </DIV>
<DIV>ln(A) = (sigma)(r^2) + ln(A1). I obtained the constant ln(A1) from
the WinNonlin program. </DIV>
<DIV> </DIV>
<DIV>My question is about which value of sigma I should use. I can use
the value of sigma obtained from Nonlin, which is based on the data.
Alternatively, I can use the sigma that Sednterp gives me, based on the
peptide composition and buffers. When making these graphs however, the
data fits exactly to the line for a trimer which uses the Nonlin sigma, and
doesn't exactly follow the slope of the line that uses the Sednterp
sigma. I understand that the sigma obtained from Nonlin comes from the
data, and perhaps that is why it fits to the data well. On the other
hand, the sigma from Sednterp is calculated using density and partial specific
volumes that are close estimates, but aren't exact. (at least this is my
understanding so far...) So I am wondering which value of sigma is
appropriate to use.</DIV>
<DIV> </DIV>
<DIV>I'm not sure whether you will be able to see this at all, but I have
enclosed a PDF to show you the different results I get using the different
sigma values. In all the papers that use the same peptide, people report
graphs very similar to the lower one, which uses sigma derived from
Nonlin. The data always overlaps the line for a trimer. However,
it isn't clear how they generated those graphs. </DIV>
<DIV> </DIV>
<DIV>I would greatly appreciate any advice you have.</DIV>
<DIV> </DIV>
<DIV>Thanks for your help,</DIV>
<DIV> </DIV>
<DIV>Tara Suntoke</DIV></BLOCKQUOTE></BODY></HTML>