[RASMB] Sedimentation equilibrium question

[Borries Demeler]

Hello Sarah,

I have always found it a lot easier to look at composition of an
unknown sample by starting off with a velocity experiment. There are
lots of analysis methods available that can ascertain homogeneity, or,
if you have heterogeneity, give you a better idea about the extent of
the heterogeneity, and indicate much more convincingly the presence of
aggregates than equilibrium ever could.

In addition, interference is quite sensitive to focussing errors, and
should your machine be slightly out of focus you would have a hard time
trying to find out why your equilibrium data doesn't fit. Fitting of 
velocity data is less sensitive to these errors.

I would recommend to run a couple of concentrations in velocity mode
(perhaps a high concentration interference experiment and a low concentration
sample measured at 230 nm - buffer should be transparent at this wavelength)
and then to look at both data sets with van Holde - Weischet analysis and
compare the integral distribution plots.

This will first tell you the extent of heterogeneity, and then allow
you to determine quite intuitively if your sample is reversibly self-
associating over the concentration range that you examined.

Feel free to contact me off line if you have additional questions.

Good luck, -Borries

> 
> 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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