From: Borries Demeler <demeler@biochem.uthscsa.edu>
  To  : Marina Mapelli , rasmb@bbri.harvard.edu
  Date: Mon, 12 Apr 1999 19:22:20 +0100

Re: ultracentrifuge data analysis

> Dear collegues,
> I'm currently working on the characterization of a single-stranded dna
> binding protein. I'm trying to understand the
> behaviour in solution of  different mutats using analytical
> ultracentrifugation. My equilibrium experiments result in a monodisperse
> preparations but the mono fitting leads to  molecular
> weights too small comapared with the ones predicted by the sequence and
> observed in denaturing gel runs (85-90 KDa instead of 120 KDa).
> My experiments take place in a buffer containing 20% glycerol and 300mM
> NaBr. I measured the density of the medium, but I'm wondering whether and
> how the partial volumes of the protein can be affected by the buffer. Or
> whether there's another explanations for this behaviour.
>  
> I also noticed that sometime I got a good fit with a biexponential fitting
> but with Mw that are one too small and one too big. Does this means that
>  protein aggregation is taking place so that the bimodal fit
> is not valid (I know that protein-protein intercations exist)?
> 

Dear Marina,

I am not sure about the reported MW, but I suspect you are using an
inappropriate model for your equilibrium fitting.

I strongly suggest to do a velocity run first to establish homogeneity/
heterogeneity. If you feel that there may be concentration dependent
association going on, it is also wise to run 3 different velocity
experiments at 3 markedly different concentrations (maybe at 210, 230 and
280 nm, or even higher). Then analyze the data with the van Holde-Weischet
method and compare the distributions. This will unambiguously tell you:

a. if there is the slightest bit of heterogeneity present 
b. if there is concentration dependent association going on
c. in case of dimerization, if the binding is tight or not.

For optimum resolution on heterogeneity in S, run as fast as possible
(60 krpm) and scan as fast as possible (delay between scans = 0,
continuous mode), and only scan a single cell at a time.

Once you established the above, you can select with higher confidence 
the appropriate model for your equilibrium fitting.

Let me know if you have additional questions.

Regards, -Borries

P.S. Check with Hans van der Zandt at your institution for help with vHW.

*******************************************************************************
* Borries Demeler                                                             *
* The University of Texas Health Science Center at San Antonio                *
* Dept. of Biochemistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760 *
* Voice: 210-567-6592, Fax: 210-567-4575, Email: demeler@biochem.uthscsa.edu  *
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