[RASMB] Sedimentation equilibrium question

[Sarah Siegel]

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