<HTML>
<HEAD>
<TITLE>Re: [RASMB] time to reach equilibrium</TITLE>
</HEAD>
<BODY>
<BLOCKQUOTE><FONT COLOR="#800000">Hi Walter (and everyone)<BR>
<BR>
Excellent - we will give WinMatch a go.<BR>
<BR>
Arthur<BR>
</FONT></BLOCKQUOTE><BR>
<BLOCKQUOTE>Dear Arthur et alias,<BR>
<BR>
Such a application does indeed exist. Although it doesn't run continuously, it can be run at anytime to check for equilibrium. It is called WinMatch (a Windows version of an even earlier program called Match). WinMatch is available from the National Analytical Ultracentrifugation Facility (NAUF) at the University if Connecticut at http://vm.uconn.edu/~wwwbiotc/AUFFTP.HTML<BR>
<BR>
WinMatch essentially performs least squares fits of a series of scans (usually taken every hour) against the last scan taken (the reference scan) and fits for horizontal (precession and vibrations) and vertical (vibrations and other variations in baseline) offsets. The result is a plot of the RMS deviation vs. time. This curve looks a lot like an exponential decay which tends toward zero slope as equilibrium is reached. It is quite useful for identifying systems that fail to reach equilibrium.<BR>
<BR>
Although calculations are handy for planning an experiment, it is the empirical determination of attainment of equilibrium that should be the deciding factor. WinMatch works very well and has been available from the NAUF for many years now.<BR>
<BR>
Walter<BR>
<BR>
<BR>
At 5:31 PM +0100 9/29/04, Arthur Rowe wrote:<BR>
<BLOCKQUOTE>Perhaps someone could write a nice little app which would continuously monitor at (say) hourly intervals the shift in fringe position at a radial position near to the base of the column, and report back to the screen when this shift either<BR>
<BR>
(1) went down to below noise level, or<BR>
(2) failed to do so (i.e. system in some sort of steady state, rather than attaining equilibrium) ?<BR>
<BR>
Just to make it nice and easy for folks . . .<BR>
<BR>
Arthur<BR>
--<BR>
*************************<BR>
Arthur Rowe<BR>
Lab at Sutton Bonington<BR>
tel: +44 115 951 6156<BR>
fax: +44 115 951 6157<BR>
*************************<BR>
<BLOCKQUOTE><BR>
<B>From:</B> "John Correia" <jcorreia@biochem.umsmed.edu><BR>
<B>Date:</B> Wed, 29 Sep 2004 10:31:11 -0500<BR>
<B>To:</B> <strauss@fmp-berlin.de>, <rasmb@server1.bbri.org><BR>
<B>Subject:</B> Re: [RASMB] time to reach equilibrium<BR>
</BLOCKQUOTE><BR>
<BLOCKQUOTE>Cheers<BR>
<BR>
We published a paper in 1977 that predicted the equilibrium distribution from earlier scans:<FONT SIZE="1"> J.J. CORREIA, G.H. Weiss and D.A. Yphantis (1977). "An Extrapolation Method for Reducing Equilibrium Times in Sedimentation Equilibrium Systems," 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). <BR>
</FONT><BR>
<FONT SIZE="1">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<B> must</B> also come from Sed velocity - Sedanal, Sedphat, etc make this very inviting. In our lab sed velocity rules, ie we have no alternative!<BR>
</FONT><BR>
<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: jcorreia@biochem.umsmed.edu <BR>
homepage location: http://biochemistry.umc.edu/correia.html<BR>
dept homepage location: http://biochemistry.umc.edu/<BR>
-------------------------------------------------------------------<BR>
<BR>
</FONT></BLOCKQUOTE><BR>
<BR>
This message has been scanned but we cannot guarantee that it and any attachments are free from viruses or other damaging content: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.<BR>
</BLOCKQUOTE><BR>
<BR>
-- <BR>
<FONT COLOR="#0000FF">----------------------------------------------------------<BR>
Walter Stafford<BR>
mailto:stafford@bbri.org<BR>
direct dial: 617-658-7808<BR>
receptionist: 617-658-7700<BR>
----------------------------------------------------------<BR>
</FONT><BR>
</BLOCKQUOTE><BR>
</BODY>
<br/>
<p>
This message has been scanned but we cannot guarantee that it and any
attachments are free from viruses or other damaging content: you are
advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.
</p>
</HTML>