Experiments
Two types of analytical centrifugation experiments exist: sedimentation velocity and sedimentation equilibrium, which are very complementary:
Sedimentation velocity
This is a sedimentation experiment which measures how fast the macromolecules move in response to centrifugal force. The sedimentation boundary movement gives us the information about mass and shape of macromolecules.
Sedimentation velocity experiment is performed at high speed rotation (30,000
- 50,000 rpm) and this type of experiment can determine the following:
- sample homogeneity (or heterogeneity) in protein mass and conformation,
- sedimentation coefficients of solute,
- presence of aggregates and their size distribution,
- overall shape/assymetry of the macromolecules,
- molecular mass and stoichiometry.
Sedimentation equilibrium
This is a sedimentation
experiment based on the balancing of centrifugal force and diffusion of the
particle in order to form a solute gradient which remains constant for an indefinite
period of time. This experiment is usually performed at a lower rotor speed
and takes several days to complete. This technique allows determination of the
following:
- molecular mass/subunit stochiometry,
- dissociation constant (KD),
- second virial coefficient (B) - the measure of nonideality of the solute (resulted from protein shape or charge).
