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<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff
size=2>David,</FONT></SPAN></DIV>
<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff size=2>It is
hard to comment without knowing more about the experiment or seeing the data.
Can you at least tell us whether you are talking about absorbance or
interference data? </FONT></SPAN></DIV>
<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff size=2>You
mention that you have considered non-ideality, which suggests that this could be
an experiment at high protein concentration. Is that correct? If so, then part
of the problem might be optical distortions due to refraction by the
boundary---these are always worst early in the run, and go away as diffusion
makes the gradient less steep. That problem can be significantly reduced by
using 3 mm centerpieces.</FONT></SPAN></DIV>
<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=838072621-24032006><FONT face=Arial color=#0000ff
size=2>John</FONT></SPAN></DIV></DIV>
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<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left><FONT
face=Tahoma size=2>-----Original Message-----<BR><B>From:</B>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <B>On
Behalf Of </B>David Hayes<BR><B>Sent:</B> Friday, March 24, 2006 12:41
PM<BR><B>To:</B> RASMB<BR><B>Subject:</B> [RASMB] Sedimentation Velocity
Experiments that are hard to fit<BR><BR></FONT></DIV>Hi to all experienced
Ultracentrifuge Scientists,<BR><BR>I have some data (repeated experiments)
from sample A buffer 1 (which may or may not have been produced on the planet
earth -- that is all I can say about it)<BR><BR>There are two puzzling aspects
to this data.<BR>First, I am not getting good repeatability in the amount of
aggregate.<BR>Second, by eye, the raw data looks like a single species (which
it should be) with a small amount of higher weight aggregate (not surprising),
but the entire data set never fits well no matter what I try.<BR>Thinking that
the repeatability problem might be an artifact of my inexperience using Sedfit
I tried many different strategies: including floating or not floating
just about every parameter and also using the nifty "experimental initial
distribution" option of Sedfit.<BR> Fitting to the whole data set, I got
a fit with a single large peak and two very small aggregate bumps which all
together integrate to about 4% of the total. The rmsd was a bit large at
0.01 and the residuals were systematic. Early scans were fit very poorly
and later scans fit somewhat poorly with scans in the middle fit pretty
well. Later using Sedfit with the single species model and only the
later half of the scans and floating a lot of things and turning off the hat
function in Sedfit brought me down to a comparable fit with a rmsd of .006,
but the residuals were still systematic and the molecular weight was off more
than what I expected for having 4% aggregate.<BR>Then I did something that
Peter Shuck thinks is a bit strange, but I think it helped me understand the
data a bit more. I fit pairs of scans with c(s) taking scans at
different times paired with the last scan. Essentially then, I was
fitting each scan individually: using the last scan in ever pair was
needed to help Sedfit know the baseline. Peter reminded me that by
fitting each scan, I lose the ability to distinguish heterogeneity from
diffusion. But in this case, all the evidence points to a single
species, so I did not think heterogeneity (beyond the measured 4% aggregation)
was significant. The peak S value stays constant all the way down the
cell. Each individual scan fit this way fit very well without systematic
residuals and a rmsd of about .005. What changed was that early scans
fit to an f/f0 of 1.06 while by the later scans f/f0 fit to 1.5. This
means that the early scans are broader than they should be for a reasonable
f/f0 and that the later scans seem to be narrower and diffusing less.
<BR>I repeated this with Sedanal dcdt based curve fitting (here I took scans
by twos going down the cell and fit to a single species ignoring the 4%
aggregate) and found a similar trend: the fitted molecular weight went
from 95,000 to 225,000 and then settled down to about 160,000. If there
were heterogeneity, the scans would fit the opposite way to a one species
model, the f/f0 would go down, or the weight would go up later in the run as
the species sedimented apart.<BR><BR>I had some interference data of
Tropomyosin that I subjected to a similar type of analysis with Sedanal,
fitting the whole data set and then fitting scans by sets of 10 for the
different times in the experiment (because of TI, RI noise fitting, it is not
possible to fit small sets of scans with Sedfit using interference data;
however, a Sedfit fit to the whole Tm data set showed no systematic variation
of the residuals dependent on scan number). There was no scan number
dependent trend in molecular weight with this molecule, though the variability
in fitted molecular weight was surprising (weights from different sets of 10
varied from 60,000 to 80,000 randomly).<BR><BR>Seeing this I tried a Sedfit
single species and Sedanal single species fits with non-ideality parameters,
but floating these parameters made no substantial difference.<BR><BR>I don't
know if anyone has run into this type of problem, but I am out of ideas what
to try next. I am being a bit stubborn wanting a good fit to the
data: the S value of the main peak is reproducible and the data contains
quite a bit of information, but I can't figure out exactly what is going
on: <BR><BR>Is this just non-ideality that I am not fitting properly or
maybe that is not modeled well?<BR>Is the culprit convection? Thoughts
on convection in the next message.<BR><BR>Cheers<BR><BR><BR><X-SIGSEP>
<P></X-SIGSEP>Dr. David B Hayes<BR>Boston Biomedical Research Institute<BR>64
Grove St.<BR>Watertown, MA 02472<BR>617-658-7738<BR><BR>
<DIV align=center><B>Boston Biomedical Research Institute... Today's Research
for Tomorrow's Health.<X-TAB> </X-TAB><BR><I>Please visit us at <A
href="http://www.bbri.org/" eudora="autourl">www.bbri.org</A></B></I>
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