From: Jo Butler <pjgb@mrc-lmb.cam.ac.uk>
  To  : Marina Mapelli , rasmb@bbri.harvard.edu
  Date: Mon, 12 Apr 1999 17:51:20 +0100

Re: ultracentrifuge data analysis

Dear Marina,

You do not say what value you are using for v-bar when trying to fit the
data.  If there is any specific hydration of the DNA (a rather likely
event), you certainly will get a marked deviation of the apparent value
away from that for DNA in pure water.  Given that your solvent is 20%
glycerol, this will probably result in an increased buoyancy of the DNA,
with the observed apparently low molecular mass.

I do not know whether anyone has measured, and published, data for apparent
partial specific volumes of DNA against glycerol concentration in the
solvent, but I am sure that this will be a significant factor.

Yours,

Jo

--On Mon, Apr 12, 1999 5:06 pm +0200 Marina Mapelli
<Marina.Mapelli@EMBL-Heidelberg.de> wrote:

> 
> 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
> 
> 
> *******************************************
>  Marina Mapelli
>  EMBL Heidelberg
>  Meyerhofstrasse n.1
>  D69012 Heidelberg
>  Germany
> 
>  e-mail:	mapelli@EMBL-Heidelberg.de 
>  tel: 	     	+49 6221 387567
>  fax: 		+49 6221 387306
> 
> *******************************************
> 



Dr P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road,
Cambridge, CB2 2QH,
UK.
Tel. 01223 248011 (or 01223 402296 DDI)
FAX. 01223 213556