[RASMB] Sedimentation equilibrium question

[Peter Schuck]

Hi Sarah,
in this situation when it's not clear what the major (and minor) species 
are, I would recommend a c(M) molar mass distribution calculated either 
from the separate equilibrium profiles or globally.  Don't know how much 
SEDFIT knowledge you have, but you can load equilibrium data into it, 
and then use the c(M) model and get the molar mass distribution.   
Refinements of the continuous molar mass distribution are available in 
SEDPHAT.
Peter


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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