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<P class=MsoNormal><FONT face=Arial>Dear Ariel and colleagues,</FONT></P>
<P class=MsoNormal><FONT face=Arial>First let me thank you for your response to
my inquiry.</FONT></P>
<P class=MsoNormal><FONT face=Arial>Maybe I am naïve, without trying to
interpret the result obtained from the equilibrium curve (John Philo is correct,
after 4days of tryptic digestion with this protein complex at 37C, some
proteolysis does occur), I was only use this equilibrium curve (or any curve),
to compare MW obtain from these two software, I thought by inputting the raw
data using exactly the same window (radius boundary), I should be getting the
same empirical number (MW), or close to it. Maybe I am off base on my
reasoning.<SPAN style="mso-spacerun: yes"> </SPAN></FONT></P>
<P class=MsoNormal><FONT face=Arial>As to solution column and time of
equilibrium issue, using the standard cell, the maximum volume one can load is
about 0.45ml. In most of my experiment, I try to shot two birds with one stone
by loading about 0.2ml to do a SV run (3-5 hours), after collecting enough scans
I then reduce the speed and reprogram for SE run (20-24 hours). I have no
problem with it if the protein is stable. By doing scan subtraction at every two
hours, I found that for 0.2ml sample, 20-24 hours is sufficient for (stable)
sample to reach equilibrium. However I found that for the radius region above
7.1, near the base of the cell, apparent due to reflections, as John Philo point
out, it will never reach zero on scan subtraction no matter how long I continue
to spin it so I try not to use the data points beyond 7.1. </FONT></P>
<P class=MsoNormal><FONT face=Arial>I agree with John Philo’s point</FONT></P>
<P class=MsoNormal><SPAN
style="COLOR: blue; FONT-FAMILY: Arial; FONT-SIZE: 10pt">“Knowing what is 'good'
or 'bad' data in equilibrium experiments is unfortunately a bit of an
art”<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office"
/><o:p></o:p></SPAN></P>
<P class=MsoNormal><FONT face=Arial><SPAN style="mso-bidi-font-size: 10.0pt">I
still remember</SPAN> a long while back, taking picture with a camera use to be
an art. One needs to know how to set f value, how to estimate distance as well
as aperture.<SPAN style="mso-spacerun: yes"> </SPAN>Expert and experience
user takes clear picture while amateur takes blur<SPAN
class=120090820-14032003>red</SPAN> picture. But now a day you can take decent
picture just with a disposable camera. Back to AUC, I was hoping I could combine
Segal and Origin to enhance my betting average of picking up the best subset to
use.</FONT></P>
<P class=MsoNormal><FONT face=Arial><SPAN
style="mso-spacerun: yes"> </SPAN>I think it is cool to be able to copy the
whole page information.<SPAN style="mso-spacerun: yes"> </SPAN>I hope
Segal Program Can provide a “page copy” function, similar to the Beckman Origin,
so page data can be copy and paste to Microsoft word for record keeping. I saw
and like the page information you sent. I could not figure out how to do
it.<SPAN style="mso-spacerun: yes"> </SPAN>I finally realize that the page
you sent is in “.jpg file”, which I believe you have to scan with Digital
camera. It would be very nice if we can just copy the page, like the Beckman
Origin does, and paste the finished page to the Microsoft word for record
keeping. At the present time I could only copy and paste each figure for record
keeping, very time consuming.</FONT></P>
<P class=MsoNormal><FONT face=Arial>I have since test another set of equilibrium
curve. It is a pure protein. It is a self-associated system.<SPAN
style="mso-spacerun: yes"> </SPAN>I am please to see these results. I also
post the raw data 00005.ra1 for anyone interest in using it for
practice.</FONT></P>
<P class=MsoNormal><FONT face=Arial>I was wondering because this curve seems
agree better with the previous curve, is this means that these software are
system dependent.<SPAN style="mso-spacerun: yes"> </SPAN>I mean if the
system is a self associated system, both software will converge to pretty much
the same answer.<SPAN style="mso-spacerun: yes"> </SPAN>If the system has
other low mass contaminate (or even some proteolysis), then the answer will not
always be the same even using the exact same window or radius region.<SPAN
style="mso-spacerun: yes"> </SPAN>This is only my wild guess.</FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P>
<P class=MsoNormal><FONT face=Arial>Regar<SPAN
class=120090820-14032003>d</SPAN>s,</FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P>
<P class=MsoNormal><FONT face=Arial>Chris<B><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt"><o:p></o:p></SPAN></B></FONT></P>
<P class=MsoNormal><FONT face=Arial><B><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt">Summary: </SPAN></B>Compare
4 edited subset data from raw data 00005.ra1 using Origin vs.. Segal.</FONT></P>
<P class=MsoHeader style="tab-stops: .5in"><FONT
face=Arial> <o:p></o:p></FONT></P>
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<TD
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<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>Edit Equilibrium curve<o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: windowtext 0.5pt solid; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 82.7pt; mso-border-left-alt: solid windowtext .5pt"
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<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>Boundary radius region
used<o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: windowtext 0.5pt solid; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 81.35pt; mso-border-left-alt: solid windowtext .5pt"
vAlign=top width=108>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>MW obtained from Origin /
variance<o:p></o:p></I></B></FONT></P>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>(Confidence limit)<o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: windowtext 0.5pt solid; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 99.25pt; mso-border-left-alt: solid windowtext .5pt"
vAlign=top width=132>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>MW obtained from Segal /
correlation<o:p></o:p></I></B></FONT></P>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial> <B><I><o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: windowtext 0.5pt solid; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 94.45pt; mso-border-left-alt: solid windowtext .5pt"
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<H3><FONT face=Arial><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt">Discrepancy@
%<o:p></o:p></SPAN></FONT></H3></TD></TR>
<TR>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: windowtext 0.5pt solid; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 85.05pt; mso-border-top-alt: solid windowtext .5pt"
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<H2><FONT face=Arial>Edit 1</FONT></H2></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 82.7pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=110>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>6.806-7.112</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 81.35pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=108>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>82KD / 11.8</FONT></P>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>(79-84KD)</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 99.25pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=132>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>82 KD / 0.999876</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 94.45pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=126>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><I><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt">0<o:p></o:p></SPAN></I></FONT></P></TD></TR>
<TR>
<TD
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vAlign=top width=113>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>Edit 2<o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 82.7pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=110>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>6.685-6.984</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 81.35pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=108>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>86KD / 4.6</FONT></P>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>(83-89KD)</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 99.25pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=132>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>87 KD / 0.999972</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 94.45pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=126>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><I><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt">-1.2<o:p></o:p></SPAN></I></FONT></P></TD></TR>
<TR>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: windowtext 0.5pt solid; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 85.05pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=113>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>Edit 3<o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 82.7pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=110>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>6.831-7.076</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 81.35pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=108>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>81KD / 11.6</FONT></P>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>(77-84KD)</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 99.25pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=132>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>83KD / 0.999018</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 94.45pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=126>
<P class=MsoNormal><FONT face=Arial><I><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt"><SPAN
style="mso-spacerun: yes">
</SPAN>-2.5<o:p></o:p></SPAN></I></FONT></P></TD></TR>
<TR>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: windowtext 0.5pt solid; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 85.05pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=113>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><B><I>Edit 4<o:p></o:p></I></B></FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 82.7pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=110>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>6.689-6.975</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 81.35pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=108>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>89KD / 4.1</FONT></P>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial>(85-92KD)</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 99.25pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=132>
<P class=MsoNormal><FONT face=Arial>89KD / 0.999959</FONT></P></TD>
<TD
style="BORDER-BOTTOM: windowtext 0.5pt solid; BORDER-LEFT: medium none; BORDER-RIGHT: windowtext 0.5pt solid; BORDER-TOP: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 5.4pt; PADDING-RIGHT: 5.4pt; PADDING-TOP: 0in; WIDTH: 94.45pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt"
vAlign=top width=126>
<P align=center class=MsoNormal style="TEXT-ALIGN: center"><FONT
face=Arial><I><SPAN
style="FONT-SIZE: 14pt; mso-bidi-font-size: 12.0pt">0<o:p></o:p></SPAN></I></FONT></P></TD></TR></TBODY></TABLE>
<P class=MsoNormal><FONT face=Arial>@ (<B><I>MW obtained from Origin<SPAN
style="mso-spacerun: yes"> </SPAN>-<SPAN style="mso-spacerun: yes">
</SPAN>MW obtained from Segal) / MW obtained from Origin
<o:p></o:p></I></B></FONT></P>
<P class=MsoNormal><FONT face=Arial><B><I>Using default rho(1.006) and
v-bar(0.73) for comparison. </I></B></FONT></P>
<P class=MsoNormal><FONT face=Arial>Use default V-bar=0.73 ,
rho=1.006</FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P>
<P class=MsoNormal><FONT face=Arial> <o:p></o:p></FONT></P></FONT></DIV>
<P><FONT face=Arial
size=2>--------------------------------------------------------------------------</FONT>
<BR><FONT face=Arial size=2>Christopher Chin</FONT> <BR><FONT face=Arial
size=2>Manager, XLA-Analytical Ultracentrifugation facility</FONT> <BR><FONT
face=Arial size=2>Sealy Center for Structural Biology</FONT> <BR><FONT
face=Arial size=2>HBC&G, 5.134 MRB.UTMB, Galveston,Tx 77555-1055</FONT>
<BR><FONT face=Arial size=2>cchin@utmb.edu, 409-772-1693, efax
708-585-1920</FONT> <BR><FONT face=Arial
size=2>---------------------------------------------------------------------------</FONT>
</P><BR>
<DIV> </DIV></BODY></HTML>