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<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2>Qin,</FONT></SPAN></DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff size=2>I
agree with Peter's response, but I wanted to point out some additional issues.
It is quite difficult to get good cancellation of the micelle signals by having
Tween in the reference buffer, particularly for absorbance studies. Most, if not
all, of the absorbance of Tween is due to impurities, and the absorbance varies
markedly depending on the manufacturer and even lot-to-lot. Unless your
reference buffer is made from the very same bottle of Tween, it is likely it
won't match the Tween in the sample. Further, Tween also makes the solutions
very good at picking up UV-absorbing extractables from plastic tubes, syringes,
etc. which creates another source of sample/reference differences. Even if
everything does match perfectly, you won't get perfect cancellation of the
micelle signals unless you match the meniscus positions in sample and reference
too.</FONT></SPAN></DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff size=2>This
has important consequences on the software side, because it is all too easy to
end up with NEGATIVE signals from the micelles (more detergent, or at least more
absorbance, on the reference side). The <EM>c(s)</EM> algorithms cannot handle
negative signals (all concentrations are forced to be positive). Thus if you do
have any negative signal (which you always will if the meniscus positions are
not matched) then the fitting algorithm cannot possibly accurately represent the
data and you will very likely get some sort of false peak. </FONT></SPAN><SPAN
class=099310522-26092005><FONT face=Arial color=#0000ff size=2>Because of these
issues I often use a buffer without the Tween as the reference sample, which
ensures the micelle signals are positive. </FONT></SPAN></DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff size=2>That 2
S value for the micelles you cited is in fact experimental, not
calculated, but as Peter said the actual micelle size varies with solvent
conditions in addition to the usual density/viscosity effects. Definitely you
should run your buffer versus water to establish the exact micelle sedimentation
coefficient for your conditions. </FONT></SPAN></DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2>Overall though when your major protein component has a sedimentation
coefficient close to that of the micelles you likely to always have significant
interference from them.</FONT></SPAN></DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2>John</FONT></SPAN></DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=099310522-26092005><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV></DIV>
<DIV><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>Qin "Chin" Zou<BR><B>Sent:</B> Wednesday, September 21, 2005 7:22
PM<BR><B>To:</B> RASMB@server1.bbri.org<BR><B>Subject:</B> [Rasmb] Tween 80
effect on c(s)<BR><BR></DIV></FONT>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">
<DIV class=Section1>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">Hi
all,<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">I wonder if anyone has
looked at the effect of Tween 80 on the s distribution. I remember that John
Philo once asked the similar question and had calculated s for Tween 80 around
2s. <o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">I have an 18KD protein
that runs at about 1.7s when there is no Tween 80 in the buffer. However, in
the presence of Tween 80, there were two species 1.2 and 1.5s (without
regularization. If F=.68, then two peaks are
overlapped).<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">The experiment was run at
60K rpm, absorbance at 276nm. Also, the fit was not good with rmsd above 0.01.
In addition, I tried to scan at 289nm, where the absorbance of Tween 80 is
minimal, with higher protein concentration (5mg/ml). Although the 1.2s peak is
not there anymore, the main peak is at 1.4s instead of the expected 1.7s. Also
the fit was quite bad with rmsd around 0.03.<SPAN
style="mso-spacerun: yes"> </SPAN>For both experiments, I had 0.02%
Tween in the reference buffer.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">Is it possible that the
1.2s peak is due to the Tween absorbance at the region between the meniscuses
of reference and sample sectors? I have not tried with the same column height
in both sectors. It is hard to match them exactly. Would it be better to run
this kind of experiment without including Tween 80 in reference buffer and
note the potential 2s peak as the Tween peak? For my protein of 1.7s, it could
be still difficult. Any input will be
appreciated.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'"><o:p> </o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">Qin "Chin"
Zou<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><SPAN class=GramE><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'">Eli Lilly and <st1:place
w:st="on">Co.</st1:place><o:p></o:p></SPAN></FONT></SPAN></P>
<P class=MsoNormal style="mso-layout-grid-align: none"><FONT
face="Courier New" size=2><SPAN
style="FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'"><o:p> </o:p></SPAN></FONT></P>
<P class=MsoNormal><FONT face=Arial size=2><SPAN
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