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<DIV><FONT size=1><FONT face=Arial><FONT
size=3>Cheers</FONT></FONT></FONT></DIV>
<DIV><FONT size=1><FONT face=Arial><FONT
size=3></FONT></FONT></FONT> </DIV>
<DIV><FONT size=1><FONT face=Arial><FONT size=3>We published a paper in 1977
that predicted the equilibrium distribution from earlier scans:</FONT>
</FONT><SPAN
style="FONT-SIZE: 11pt; LAYOUT-GRID-MODE: line; FONT-FAMILY: Arial; mso-bidi-font-size: 10.0pt; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA">J.J.
CORREIA, G.H. Weiss and D.A. Yphantis (1977). "An Extrapolation Method for
Reducing Equilibrium Times in Sedimentation Equilibrium Systems,"<SPAN
style="mso-spacerun: yes"> </SPAN>Biophysical J. 20, 153‑168. The
point being, as Holger just mentioned, many samples aggregate with time, or
display insolubility, instability. Many of us have observed this with
proteins like tubulin which like to associate in polymorphic forms, and with
many His tagged proteins. In many of these cases there is a slow loss of
material from a "stable" exponential distribution. Information can still
be extracted, with caution. Solutions to this problem have included change
the pH, ionic strength, etc (obvious), drop the temperature, add reducing agent
(TCEP has worked best in limiting cases), remove the His tag, go to a short
column format (which speeds up the time dramatically (16-fold for a 4-fold
reduction in column height). </SPAN></FONT></DIV>
<DIV><FONT size=1><SPAN
style="FONT-SIZE: 11pt; LAYOUT-GRID-MODE: line; FONT-FAMILY: Arial; mso-bidi-font-size: 10.0pt; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"></SPAN></FONT> </DIV>
<DIV><FONT size=1><SPAN
style="FONT-SIZE: 11pt; LAYOUT-GRID-MODE: line; FONT-FAMILY: Arial; mso-bidi-font-size: 10.0pt; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA">I
not only agree with Bo, I insist that you do Sed velocity before you waste time
doing sed equilibrium - I suspect most people do this - furthermore for many
systems the quantitative analysis <STRONG>must</STRONG> also come from Sed
velocity - Sedanal, Sedphat, etc make this very inviting. In our lab sed
velocity rules, ie we have no alternative!</SPAN><BR></FONT></DIV>
<DIV> </DIV>
<DIV><FONT
size=1>-------------------------------------------------------------------<BR> Dr.
John J. "Jack" Correia<BR> Department of Biochemistry<BR> University
of Mississippi Medical Center<BR> 2500 North State Street<BR> Jackson,
MS 39216<BR> (601)
984-1522
<BR> fax (601)
984-1501
<BR> email address: <A
href="mailto:jcorreia@biochem.umsmed.edu">jcorreia@biochem.umsmed.edu</A>
<BR> homepage location: <A
href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</A><BR> dept
homepage location: <A
href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</A><BR>-------------------------------------------------------------------<BR> <BR> <BR></DIV></FONT></BODY></HTML>