[RASMB] Sedimentation equilibrium question

[Tom Laue]

Hi Sarah-
Have you done sedimentation velocity analysis of your samples? What does 
it show with respect to the concentration dependence of the g(s) 
patterns? This sort of analysis will tell you whether it is worthwhile 
doing equilibrium sedimentation by: 1) showing whether there is 
aggregate, 2) showing if there is significant mass action 
association/dissociation in the concentration range where you are 
working and 3) making sure there are no smaller species.
With respect to the equilibrium analysis- fit the individual data sets 
first to a simple model (average molar masses as a function of 
concentration).Essentially doing what Jo and Ariel suggested. There are 
several programs to help with this: Sedanal has Biospin built into it, 
MStar, Segal. The trick with any of these analyses is making sure the 
signal is equal to the concentration, and it is the presence of any 
baseline offset (i.e. zero signal does not equal zero concentration) 
that is the usual concern. For absorbance data, the baseline offset 
usually is small and can be determined experimentally by pelleting your 
material (or at least making sure some data are acquired at a rotor 
speed where the meniscus region is entirely depleted of protein). 
Interference optics are tougher to deal with since the baseline offset 
may change with rotor speed (due to window stresses). You may use 
HeteroAnalysis, fitting to an ideal single component and fit for the 
baseline offset. In this latter case, M will be between the weight and 
z- average, typically closer to the latter.
See if the individual data sets reveal systematic behavior with respect 
to concentration and rotor speed. If the M increases with concentration 
(I would suggest acquiring data over a wider initial concentration 
range), then you have a mass action association, plus you have some idea 
(within a decade) of the monomer concentration range where the mass 
action association is important to protein behavior. If M decreases 
significantly with increasing rotor speed, you probably have 
irreversible aggregate (but you would know this clearly from velocity 
analysis).
Best wishes,
Tom

Sarah Siegel wrote:
> Hi Everyone,
>
> I am a beginner in the world of AUC and I'm having a lot of trouble 
> fitting sedimentation equilibrium data.  I was hoping someone could 
> help me figure out if my problem is in the way I'm fitting or the way 
> I set up my experiment.  I did attend last spring's AUC workshop at 
> UConn, but this is my first time really running an equilibrium 
> experiment.  The system I have is an 84 kDa purified protein that may 
> be in equilibrium with dimer, possibly also some larger species.  I am 
> trying to get an idea of what the dominant species present are.  I ran 
> two samples, at 1.0 and 0.5 mg/mL, collecting interference data at a 
> range of speeds from 8,000 rpm to 20,000 rpm.  I watched the approach 
> to equilibrium using Match in HeteroAnalysis.  I have been using 
> HeteroAnalysis to try to fit the data globally.  I have tried fitting 
> to several models, including ideal and nonideal systems, 
> monomer-dimer, monomer-trimer, monomer-tetramer, and several three 
> species equilibria.  So far the best fit I have has rms deviations of 
> 0.027 using a nonideal model, however this fit also tells me the 
> molecular weight of the protein is 138 kDa, which is larger than 
> monomer and smaller than dimer, and the residuals do not have a random 
> pattern.  Does anyone have any ideas about another way I should be 
> fitting this, or other data I should collect?  Thanks for your help!
>
> Sarah
>
> Sarah Siegel
> Graduate Student
> The Scripps Research Institute
> La Jolla, CA
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
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Phone: 603-862-2459
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E-mail: Tom.Laue at unh.edu
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