[RASMB] Sedimentation equilibrium question

[Jo Butler]

Dear Sarah,

I know it may seem old-fashioned, but many of my colleagues bring me 
complex systems and ask for my help, and I have always found it useful 
to get a simple plot of Mw,app against c as a first step in any analysis 
of an unknown sample.  (This is easily done by taking a sliding window 
of a suitable number of data points, calculating Mw,app from 
dln(c)/dr**2, and plotting this against the central concentration for 
the datum points.)
 From this one can usually see the nature of the aggregation, what the 
smallest species is likely to be and whether there is any major 
non-ideality.  One can then move on to model fitting with a good idea of 
what might be relevant, rather than simply trying all possible models 
and attempting to guess from the residuals what is not fitting well (let 
alone why not).

Jo

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
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb

-- 
Dr P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296