Classical experiments of Meselson and Stahl in 1957 utilised equilibrium density gradient centrifuga-
tion in caesium chloride (CsCl) to establish unambiguously the semi-conservative nature of DNA replica-
tion. A CsCl density gradient can also be established by diffusion under the influence of a centrifugal force
to give a range of densities that encompass the buoyant densities of the molecular species to be separated.
When centrifuged in such a gradient, a sample of DNA migrates until it reaches the point at which its own
buoyant density equals that of the CsCl gradient. A number of different factors affect the buoyant density of
DNA, for example the GC content, the degree of hydration, the presence of other ions, pH or the presence
of DNA binding or intercalating agents. The fact that intercalating dyes alter the buoyant density of DNA has
been exploited to provide an experimental method for the preparative purification of DNA. The concentra-
tion of ethidium bromide bound to DNA is dependent on the superhelical state of the DNA. For example
closed, circular plasmid DNA binds at a higher density than either nicked circular or linear DNA. Other cel-
lular components are removed as a pellet at the bottom of the tube (e.g.RNA) or as a surface layer (proteins).
Modern, computer controlled, AUC instruments (such as the Beckman XL-I, which is equipped with both
absorption and interference optics) allow characteristics such as molecular mass, stoichiometry of complexes,
conformation and shape, diffusion and sedimentation, self-association and equilibrium constants to be studied
quantitatively (Figure 11.12). The analytical ultracentrifuge spins the sample under a vacuum at a set spin
speed and fixed temperature. At predefined time intervals, the instrument records the concentration distri-
bution in the sample by measuring the absorbance. It is possible to attain speeds as high as 60,000rpm
(250,000 g). The sample itself is a solution in its native state and under biologically relevant conditions.
It is held in specialised cells designed to withstand the high gravitational forces, which at the same time
allow transmittance of light through the sample in the wavelength range 200–800nm. The centrifuge cells
accommodate both the sample and reference buffer, and the cells are assembled prior to each experiment
from a cell housing, quartz or sapphire windows and one of several different types of centrepieces.
Different types of rotors are available that can be used at different spin speeds or that contain either four
or eight cell housings, one of which is used for the reference buffer in each case.
In general there are two different approaches to AUC: (1) sedimentation velocityand (2) sedimentation
equilibrium. Sedimentation velocity is a hydrodynamic technique that is a probe for both mass and shape
440 Chapter 11
Figure 11.12 Schematic diagram of the optical arrangement of an analytical ultracentrifuge (this diagram is based
on the Beckman Optima XL-A)