From: Allen Minton <minton@helix.nih.gov>
  To  : Les Hicks <hicks@gpu.srv.ualberta.ca>
  Date: Sun, 14 May 2000 18:07:19 -0400

Re: Sedimentation Equilibrium of highly concentrated proteinsolutions

Reply to Les Hicks:

We have been doing sedimentation equilibrium of highly concentrated protein 
solutions for many years. Marc Lewis and I did s.e. of hemoglobin and 
myoglobin at concentrations exceeding 350 mg/ml (Minton and Lewis, Biophys. 
Chem. 14, 317; 1980).   To use absorbance optics, you have to get the light 
path way down.  We did this by the trick of using a rubber window gasket as 
the centerpiece!   You build the cell by using an old 10 mm centerpiece cut 
in half, making two ca. 5 mm centerpieces, as spacers outside of the 
windows to hold the sandwich of two windows plus gasket-centerpiece.  When 
this is tightened up you can get a path length as small as a few tenths of 
a mm.

Although it seems that nonoptical methods aren't too popular with XLA/I 
jocks, I would nevertheless like to put in a plug for our method of doing 
s.e. of labeled tracer solutes with a prep centrifuge, with subsequent 
quantitation of gradients via fractionation (for a recent description, see 
Rivas et al, Methods 19, 194; 1999).  It is a no-brainer to get the 
concentration way up simply decreasing the ratio of labeled to unlabeled 
solute.  We are very interested in characterizing associations in crowded 
media and routinely work at macromolecular concentrations up to 100, 200 or 
more mg/ml.  You can also do tracer s.e. in the XLA with a chromophorically 
labeled protein (Rivas et al, Biochemistry 38, 9379; 1999), but at high 
concentrations you have to be careful about keeping the gradient of 
concentrated solute low so you don't get stung by Wiener skewing.

Allen Minton