From: Cole, James L <jim_cole@merck.com>
  To  : 'Marina Mapelli' <Marina.Mapelli@EMBL-Heidelberg.de>
  Date: Mon, 12 Apr 1999 13:03:08 -0400

RE: ultracentrifuge data analysis

Dear Marina:

Protein V-bar is affected by glycerol  [Gekko and Timasheff (1981)
Biochemistry, 20,4667-4676].  This  paper,  demonstrates  that v-bar
increases with glycerol concentration, and the effect is similar for four
proteins.  From this data,  we calculated a correction factor:
Delta vbar = +3.33 X 10-4 / % volume glycerol.  

For a typical  protein and buffer system,   this correction increases the
calculated molecular mass by less  than 5% for 20% glycerol content, so
something else must be going on in your experiment. I agree with Bo Demeler
that it would be a good idea to check the homogeneity of the sample with a
velocity run.

Jim
----------------------

> 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)?
> 
> 
> many thanks in advance
> best regards
> marina
> 
> 
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>  Marina Mapelli
>  EMBL Heidelberg
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> 
>  e-mail:	mapelli@EMBL-Heidelberg.de 
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