[RASMB] sedimentation

[Peter Schuck]

Irene,

I also disagree with Kristian about the upper size limit.  I would argue 
there is no such thing (except perhaps what geometrically fits into the 
cell):  Particles can be quite different and their behavior in the AUC 
depends on the experimental conditions (including buffer density, which can 
be used to reduce the sedimentation).

I have run small bacteria in the AUC to determine their 
size-distribution.  We know that they cannot swim as fast as they sediment, 
and I think my collaborators starved them so that they wouldn't do anything 
funny.  Average size was 6 micrometer, or 30 GDa (by STEM 
electronmicroscopy).  We simply sedimented a dilute solution at 3000 rpm 
(in ordinary phosphate buffer) and used the interference optics to 
scan.  Didn't encounter any of the detection problems Kristian mentioned - 
this is probably dependent on the nature of the particles and the 
concentration you use.  Our loading concentration was in the ballpark of 
0.3 fringes.  That's plenty for a sedimentation velocity analysis with 
sedfit.  Roughly estimating the results, dividing the peak sedimentation 
coefficient (10,000 S) by the average diffusion coefficient, which was 
measured separately by dynamic light scattering, gives a molar mass value 
that is in the right ballpark.  The distribution started at a couple of 
hundred S, showing a strong peak at 10,000 S, but tailed up to 100,000 S, 
suggesting that some of the bacteria have grown much larger than 6 
micrometers or have associated, or rather socialized, which they are known 
to do.

 From this it appears that there is not necessarily anything going terribly 
wrong or creating unusual difficulties when dealing with very large 
particles, under dilute conditions and very low rotor speeds.   It may be 
different with your particles, but I would just try.

Good luck,
Peter