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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'>Thank you all for your input. That did
help clear out many of my thoughts.<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'>First, to answer Tom’s questions, I
did not have chances to do the experiments you suggested. And I try not to
disturb my samples too much.<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'>Peter and John suggested not including
Tween in reference buffer. This may be a good idea to make things more straightforward.
Thanks. Also at 5mg/ml, the fits were bad, which may indicate the non-ideality.
<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'>As far as the Tween 80 absorbance, I did
scan the buffer from 290-230nm in UV spec and there are some absorbance between
285-260nm and it then really takes off at < 260nm. This is at Tween
concentration of 0.02%.<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>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Now I would like to further ask if it is possible
to just look at the ratio such as 280nm/250nm since for Tween this ratio could
be quite small while there are still enough signals for the protein. I would
think to do this at protein concentration around 1-2mg/ml (this protein has an extinction
coefficient of 0.55). Thanks.<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>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Chin<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>
<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><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'>
rasmb-admin@server1.bbri.org [mailto:rasmb-admin@server1.bbri.org] <b><span
style='font-weight:bold'>On Behalf Of </span></b>Qin "Chin" Zou<br>
<b><span style='font-weight:bold'>Sent:</span></b> Wednesday, September 21,
2005 9:22 PM<br>
<b><span style='font-weight:bold'>To:</span></b> RASMB@server1.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> [Rasmb] Tween 80 effect
on c(s)</span></font><o:p></o:p></p>
</div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;font-family:"Courier New"'>Hi
all,<o:p></o:p></span></font></p>
<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;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;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;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;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;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;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;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;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;font-family:"Courier New"'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;font-family:"Courier New"'>Qin
"Chin" Zou<o:p></o:p></span></font></p>
<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;font-family:"Courier New"'>Eli
Lilly and <st1:place w:st="on">Co.</st1:place><o:p></o:p></span></font></p>
<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 face="Courier New"><span style='font-size:10.0pt;font-family:"Courier New"'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'><o:p> </o:p></span></font></p>
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