Analyt Ultracent 3

Actual analytical ultracentrifugation output

661 University Avenue, MaRS Centre, West Tower, 16th Floor
Centrifuge operator: Elisa Leung
Principle Investigator: Dr. Walid A. Houry
Email: elisa.leung@utoronto.ca
TEL (416) 946-7364
FAX (416) 978-8548

 

What information can we currently obtain from sedimentation analysis?

There are two fundamental sedimentation experiments: Sedimentation Equilibrium and Sedimentation Velocity. Both experiments require approximately 250 µL sample at a concentration range between 20 µg/mL and 5 mg/mL.

Sedimentation Equilibrium Experiments

  1. Determination of the molecular weight of macromolecules (in the range of 1,000 – 2,000,000 Da) in aqueous solution at a defined pH, temperature (2– 45 degC), salt, and ligand concentration.
  2. Determination of the stoichiometry and equilibrium constants for self-association reactions. (Example: monomer < — > trimer; monomer < — > dimer < — > tetramer; etc.).
    Note: We are currently unable to determine equilibrium constants for mixed association reactions (e.g. protein A + protein B < — > protein complex AB) because of limitations in software.
  3. Determination of the stoichiometry for stable complexes of two different macromolecules.
    Example: a complex of two or more different proteins that do not dissociate at concentrations above 0.1 mg/mL.
  4. Obtain a qualitative estimate of the polydispersity of a macromolecule solution.

Sedimentation Velocity Experiments

There are several different velocity experiments that can be used to obtain information regarding polydispersity, overall hydrodynamic shape of macromolecules, sedimentation, diffusion, and frictional coefficients. In our opinion, there is one velocity experiment (developed in 1992 by W. Stafford III) that is particularly useful for modern biological applications, the sedimentation time derivative analysis. This method calculates a distribution function of sedimentation coefficients for the molecular species present in the solution. For example, if the solution contains a monomeric species in equilibrium with a large aggregate, a bimodal distribution of sedimentation coefficients would be observed, and the sedimentation coefficient of monomer and aggregate can be calculated. At present this is the best method we have for examining mixed associations and assessing polydispersity.

How to Request an Ultracentrifugation Run?

  1. Download and complete a Request Form. Click here.
  2. Read information on how to prepare sample. Click here.