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Analytical Ultracentifugation


Analytical ultracentrifugation relies on the simple premise that biomolecules in solution will sediment in the presence of the gravitational force applied by the centrifuge. The rate at which the particle sediments will be affected by its molecular weight, size and shape allowing one to determine these parameters experimentally. There are two basic types of experiments performed with the analytical ultracentrifuge, sedimentation velocity and sedimentation equilibrium.

Sedimentation velocity experiments rely on a high angular velocity which causes the solute to sediment rapidly leading to a depletion of solute near the meniscus. A boundary forms between the depleted and uniform concentration areas of the solute which can be monitored to determine the sedimentation coefficient which is a measure of the effective size of the solute.

Sedimentation equilibrium experiments employ a smaller angular velocity than sedimentation velocity which causes the solute to sediment at a much slower rate. As sedimentation occurs, diffusion opposes the gradual concentration increase in the bottom of the cell. After the two opposing forces reach equilibrium, the diffusional flow exactly balances the sedimentation flow leading to a concentration profile that is constant over time from which the molecular weight of the solute can be determined.

Applications

  • Examination of Sample Purity
  • Molecular Weight Determination
  • Analysis of Associating Systems
  • Detection of Conformational Changes
  • Ligand Binding
  • References (click at left for links)

Instrument Info:

  • XL-A (Beckman Coulter, Installed 1999)
  • Rotor: An-60 Ti - 4-place titanium rotor rated for 60,000 rpm
  • An-Ti 50 – 8-place titanium rotor rated for 50,500 rpm
  • XL-I (Beckman Coulter, Installed 2005)

Sample Requirements:

Sedimentation Velocity: At least 500uL of sample with an OD of 0.7-0.8 at the scanning wavelength. Bring at least 5 mL of buffer as well.

Sedimentation Equilibrium: At least 500uL of sample with an OD of 0.2-0.3 at the scanning wavelength. Bring at least 10mL of buffer as well.

Training

Training is administered by the staff of the biophysics core and must be completed in advance of any experiments.