Sedimentation equilibrium

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Sedimentation equilibrium is an analytical ultracentrifugation method for measuring protein molecular masses in solution and for studying protein-protein interactions. Particular applications of this technique are:

• establishing the native state of a protein as a monomer, dimer, trimer, etc.
• measuring the equilibrium constant for association of proteins which reversibly self-associate to form oligomers.
• 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.
• measuring the equilibrium constants for reversible protein-protein and protein-ligand interactions (approximate range 1 nanomolar to 1 millimolar).

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 of the molecular masses determined by this technique is usually 1-2%.

[edit] External links

• [1]
• Reversible Associations in Structural and Molecular Biology