[RASMB] Sedimentation equilibrium question

[Allen Minton]

Jo and others -

I second Jo regarding the usefulness of a plot of Mw,app vs 
concentration.  But when computing Mw,app vs a function of c, you 
have to make sure that you have an accurate baseline correction, 
particularly near the meniscus.  You can't trust the nominal (sample 
- reference) value reported by the instrument.  If you have residual 
baseline uncertainty, the value of Mw,app calculated assuming no 
baseline correction can go seriously haywire as the signal from 
solute approaches the baseline.  I recommend a high speed meniscus 
depletion following the equilibrium scan, then returning the rotor to 
the original speed and rescanning to measure signal in the depleted 
meniscus region.

Allen Minton


At 07:28 AM 3/29/2007, Jo Butler wrote:
>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
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>
>--
>Dr P.J.G. Butler,
>MRC Laboratory of Molecular Biology,
>Hills Road, Cambridge, CB2 2QH, UK.
>Tel. +44 (0)1223 402296
>
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