The Technique

Analytical ultracentrifugation is the study of the behaviour of macromolecules in solution analysed under the influence of a strong gravitational force. Most macromolecules have a different density from the solvent surrounding them and by analysing while centriuging will sink (or float) in a strong enough field. Observations of dynamic behaviour ("sedimentation velocity") or of systems in equilibrium ("sedimentation equilibrium") can provide information about size, shape, density and conformational changes in proteins and other macromolecules. The technique can be applied to materials ranging in size from peptides to viruses and living cells. 

Applications

  • Determine of protein homogeneity in solution (or a mixture of forms, e.g. monomer/dimer or aggregates).
  • Determine of conformational changes associated with oligomerisation or binding of another component
  • Determine of molecular weights or subunit stoichiometry (monomer, dimer, trimer etc) in solution using sedimentation equilibrium. The analytical ultracentrifuge provides information about the oligomeric state of a protein and is more accurate than gel filtration.

Facilities Available

Beckman Optima XL/I (Room 409 Molecular Biosciences Building). This machine is fitted with an absorption optical system capable of wavelengths between 200 and 750 nm (A values between 0.1 and 1.4) and the Rayleigh ("interference") system, which uses laser light to measure refractive index changes and is well suited to high protein concentrations (over 2 mg/ml) and study of substances without usable chromophores (e.g. polysaccharides).
Although experiments are normally run at 20°C, the machine is capable of thermostatting from 4°C to over 50°C.

Sample Requirements

Velocity experiments:  ~450 ul sample required ideally with absorption ~0.8-1 at the wavelength to be used.  Up to three samples may be run at once.

Equilibrium experiments:  ~30 ul per sample, with absorption about 0.4.  Up to nine samples can be run at once (eg, different concentrations or conditions).

In both cases an equal volume of reference buffer per sample is necessary, identical to the sample buffer (eg, for dialysed samples, use the buffer from outside the dialysis bag).  These are only guidelines - expect to discuss sample preparation in detail with the user before beginning a run.

Charges for use

•    Velocity run $100
•    Equilibrium run $200

UQ users have priority of access. Note: researchers will need to undertake the relevant building and other inductions prior to using equipment in the facility without supervision.

Contact

Dr Amanda Nouwens
a.nouwens@uq.edu.au
334609490

 

 

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