measuring the stoichiometry of complexes between two or more different proteins (e.g. a soluble receptor and its ligand or an antigen-antibody pair), or between a protein and a non-protein ligand.
In sedimentation equilibrium the sample is spun in an analytical ultracentrifuge at a speed high enough to force the protein toward the outside of the rotor, but not high enough to cause the sample to form a pellet. As the centrifugal force produces a gradient in protein concentration across the centrifuge cell, diffusion acts to oppose this concentration gradient. Eventually an exact balance is reached between sedimentation and diffusion, and the concentration distribution reaches an equilibrium. This equilibrium concentration distribution across the cell is then measured while the sample is spinning, using either absorbance or refractive index detection. The key point about sedimentation equilibrium is that the concentration distribution at equilibrium depends only on molecular mass, and is entirely independent of the shape of the molecule. The precision aberration of the molecular masses determined by this technique is usually 1-2%.
