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<TITLE>Re: [RASMB] Sedimentation equilibrium question</TITLE>
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Hi Sarah<BR>
<BR>
There are lots of good suggestion coming your way - so I will not duplicate. But one little point does occur to me. You sound to be floating the (reduced) molecular weight of your protein. Assuming that what you have is a chemically pure system (i.e. there is only one covalently bound entity present (you say it is 84 kDa, purified) then you should be <I>fixing</I> this parameter for the monomeric species, whatever mode of analysis you use.<BR>
<BR>
Oh, and one little thing more. There are dangers in the "try every model you can think of, select the one which has best residuals/whatever as being <U>correct</U>" approach. It has long been appreciated that simple theory shows that if you keep on doing this, then you will always, eventually, come up with something that looks good. Doesn't mean it's right, though. The famous example of this is the search (a few years back) for ways of predicting earthquakes - which eventually came up* with a statistically good correlation between occurrence of terrestrial earthquakes and the phases of a moon of the planet Uranus!<BR>
<BR>
Kind regards<BR>
<BR>
Arthur<BR>
<FONT SIZE="2">*the authors <I>did</I> report this with tongue firmly in cheek, I should add.<BR>
</FONT>- <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>Tom Laue <tml@cisunix.unh.edu><BR>
<B>Organization: </B>CAMIS-UNH<BR>
<B>Reply-To: </B>Tom.Laue@unh.edu<BR>
<B>Date: </B>Thu, 29 Mar 2007 09:06:47 -0400<BR>
<B>To: </B>Sarah Siegel <ssiegel@scripps.edu><BR>
<B>Cc: </B>rasmb@server1.bbri.org<BR>
<B>Subject: </B>Re: [RASMB] Sedimentation equilibrium question<BR>
<BR>
</BLOCKQUOTE><BR>
<BLOCKQUOTE><TT>Hi Sarah-<BR>
Have you done sedimentation velocity analysis of your samples? What does <BR>
it show with respect to the concentration dependence of the g(s) <BR>
patterns? This sort of analysis will tell you whether it is worthwhile <BR>
doing equilibrium sedimentation by: 1) showing whether there is <BR>
aggregate, 2) showing if there is significant mass action <BR>
association/dissociation in the concentration range where you are <BR>
working and 3) making sure there are no smaller species.<BR>
With respect to the equilibrium analysis- fit the individual data sets <BR>
first to a simple model (average molar masses as a function of <BR>
concentration).Essentially doing what Jo and Ariel suggested. There are <BR>
several programs to help with this: Sedanal has Biospin built into it, <BR>
MStar, Segal. The trick with any of these analyses is making sure the <BR>
signal is equal to the concentration, and it is the presence of any <BR>
baseline offset (i.e. zero signal does not equal zero concentration) <BR>
that is the usual concern. For absorbance data, the baseline offset <BR>
usually is small and can be determined experimentally by pelleting your <BR>
material (or at least making sure some data are acquired at a rotor <BR>
speed where the meniscus region is entirely depleted of protein). <BR>
Interference optics are tougher to deal with since the baseline offset <BR>
may change with rotor speed (due to window stresses). You may use <BR>
HeteroAnalysis, fitting to an ideal single component and fit for the <BR>
baseline offset. In this latter case, M will be between the weight and <BR>
z- average, typically closer to the latter.<BR>
See if the individual data sets reveal systematic behavior with respect <BR>
to concentration and rotor speed. If the M increases with concentration <BR>
(I would suggest acquiring data over a wider initial concentration <BR>
range), then you have a mass action association, plus you have some idea <BR>
(within a decade) of the monomer concentration range where the mass <BR>
action association is important to protein behavior. If M decreases <BR>
significantly with increasing rotor speed, you probably have <BR>
irreversible aggregate (but you would know this clearly from velocity <BR>
analysis).<BR>
Best wishes,<BR>
Tom<BR>
<BR>
Sarah Siegel wrote:<BR>
> Hi Everyone,<BR>
><BR>
> I am a beginner in the world of AUC and I'm having a lot of trouble <BR>
> fitting sedimentation equilibrium data. I was hoping someone could <BR>
> help me figure out if my problem is in the way I'm fitting or the way <BR>
> I set up my experiment. I did attend last spring's AUC workshop at <BR>
> UConn, but this is my first time really running an equilibrium <BR>
> experiment. The system I have is an 84 kDa purified protein that may <BR>
> be in equilibrium with dimer, possibly also some larger species. I am <BR>
> trying to get an idea of what the dominant species present are. I ran <BR>
> two samples, at 1.0 and 0.5 mg/mL, collecting interference data at a <BR>
> range of speeds from 8,000 rpm to 20,000 rpm. I watched the approach <BR>
> to equilibrium using Match in HeteroAnalysis. I have been using <BR>
> HeteroAnalysis to try to fit the data globally. I have tried fitting <BR>
> to several models, including ideal and nonideal systems, <BR>
> monomer-dimer, monomer-trimer, monomer-tetramer, and several three <BR>
> species equilibria. So far the best fit I have has rms deviations of <BR>
> 0.027 using a nonideal model, however this fit also tells me the <BR>
> molecular weight of the protein is 138 kDa, which is larger than <BR>
> monomer and smaller than dimer, and the residuals do not have a random <BR>
> pattern. Does anyone have any ideas about another way I should be <BR>
> fitting this, or other data I should collect? Thanks for your help!<BR>
><BR>
> Sarah<BR>
><BR>
> Sarah Siegel<BR>
> Graduate Student<BR>
> The Scripps Research Institute<BR>
> La Jolla, CA<BR>
> _______________________________________________<BR>
> RASMB mailing list<BR>
> RASMB@rasmb.bbri.org<BR>
> http://rasmb.bbri.org/mailman/listinfo/rasmb<BR>
><BR>
<BR>
-- <BR>
Department of Biochemistry and Molecular Biology<BR>
University of New Hampshire<BR>
Durham, NH 03824-3544<BR>
Phone: 603-862-2459<BR>
FAX: 603-862-0031<BR>
E-mail: Tom.Laue@unh.edu<BR>
www.bitc.unh.edu<BR>
www.camis.unh.edu<BR>
<BR>
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