[RASMB] Sedimentation equilibrium question

[Leech, AP]

Hi Sarah,

I don't think I can add significantly to the helpful advice already
given, but on a slightly different tack I found it is a real
confidence booster if you can find a nice, well behaved, stable
protein and do some equilibrium runs on it, if possible over a wide
range of speeds and concentrations, even different wavelengths. This
will give you a good idea of when you can trust the data the machine
gives you, and what the analysis programs make of it, and when you
should take it with a pinch of salt. It will also give you a better
idea of what the residuals look like in a simple system.

The other thing I would say is that with a complex system it is
sometimes better to go back to sample prep and conditions if these
can make things simpler, and then do a new experiment, rather than
trying to squeeze too much out of a single data set. Sometimes
people who bring proteins have an overly optimistic idea of purity
and homogeneity!

Hope this is useful,

Andrew

PS The great thing about the world of AUC is that even as a beginner
you can post a query to the RASMB and get friendly advice from real
experts in the field!

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 Andrew Leech                   *  Laboratory Manager
Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
PO Box 373,  York  YO10 5YW       *  Email : apl3 at york.ac.uk