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Analytical Ultracentrifugation

Equipment available: Beckman Optima XL-I analytical ultracentrifuges
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £85/day (24 hr period)

 

Analytical ultracentrifugation

Analytical ultracentrifugation (AUC) is the study of the behaviour of macromolecules in solution under the influence of a sedimentation force, generated by placing a sample in a spinning rotor. 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. This is normally run as a service by the facility manager with samples supplied by the users although regular users can be trained to use the instrument by themselves.

Applications

The main use of sedimentation velocity experiments is to examine whether a protein sample is homogeneous in solution or a mixture of forms (e.g. monomer/dimer, or aggregates). It may be possible to tell whether its shape changes significantly or if association with another component in the solution is taking place. It also has an enormous dynamic range i.e. in the same sample one can observe a single protein and high MW complex e.g. ribosome.

Sedimentation equilibrium is particularly useful for determination of molecular weights or subunit stoichiometry (monomer, dimer, trimer etc.) in solution.

Facilities

Beckman Optima XL-I

This instrument 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). The interference data is used in preference to absorbance due to its better signal to noise.

The centrifuge will run at speeds up to 60000 rpm, though currently we only use a maximum of 40000 rpm for equilibrium and velocity experiments.

The cells are composed of centrepieces and quartz or sapphire windows, assembled in an aluminium housing. The rotors are of titanium, and run in a vacuum for best temperature stability. Although experiments are normally run at 20°C, the machine is capable of thermostatting from 5°C to over 50°C.

Rotors & cells

We have an AN60Ti four hole, capable of 60000 rpm. All AUC runs require use of a single counterbalance cell for calibration purposes, which limits the maximum number of cells to three.

There are three double sector velocity cells that are used for both velocity and equilibrium experiments. The centrepieces are made of charcoal filled Epon (an epoxy resin) and come in a standard 12mm light path and also a 3mm form for working with smaller volumes. 12mm aluminium centrepieces for higher speeds are also available. The optical windows are quartz or sapphire. Sapphire has better mechanical properties for work at high speeds but quartz has better UV transmission at low wavelengths (<240 nm).

Each cell has sample compartments arranged in pairs for sample and reference - the optical measurements are always taken as the difference between these two to minimise optical and other artefacts. This means that for each sample a suitable reference buffer is required.

Samples

For velocity experiments 400 µl sample is required ideally with absorption 0.8-1 at the wavelength to be used. Equilibrium experiments need 100 µl per sample, with absorption above 0.1. In both cases an equal volume of reference buffer per sample is necessary.

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Page Last Updated: 26/03/2015 by Dr D. Staunton
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