From: Joachim Behlke <behlke@orion.rz.mdc-berlin.de>
  To  : RASMB@bbri.eri.harvard.edu
  Date: Wed, 27 Apr 1994 18:38:09 +0000

XL-A absorption optics

Hi, as a newcomer in XL-A technique I have to learn something what I have not
observed in such extent with the Model E using absorption or Rayleigh inter-
ference optics. For the experiments of heterologous association (different
proteins or protein and nucleic acid) we need exact information about the
molecular mass of the reactants under the choosen conditions and also the
initial concentrations. In sedimentation equilibrium runs with reactants of
different molecular masses we have to find a useful speed, initial
concentration or wave length where we can record a suitable concentration
or absorption distribution. In some experiments we have used wave lengths
lower than 240 nm to get a good signal/noise ratio. Sometimes, depending on
speed and initial concentration at the bottom of the cell we obtained values
higher than 1 or 2. Considering these data the molecular mass of the
proteins was lower than expected but when considering the virial coefficient
(a positive value) we get the nearly expected molecular mass. This is
surprising for me because we have used very low concentrations of 0.1-0.2
mg/ml. Calculation of molecular mass of the same sample from the
distribution at other wave lengths with lower absorption yield normal
data. From these observations I would like to ask the XL-A user for similar
experience or to know until to which absorption (0.8, 1.0 or somewhat
higher) the Lambert law is valid for the concentration determination in XL-A?
Best regards.
Joachim Behlke