The Departmental analytical ultracentrifugation facility is equipped with two analytical ultracentrifuges, an Optima XL-A with absorbance optics and an Optima XL-I with absorbance and interference optics.  The centrifuges are housed in the Glycobiology Institute and are available for use by members of the Department of Biochemistry.

Absorbance optics

Both centrifuges have absorbance optics which allow the use of wavelengths between 190 and 800 nm.  They can perform wavelength scans at a fixed position in the sample cell and radial scans at a fixed wavelength across the cell.  The absorbance optics can be used for sedimentation velocity and sedimentation equilibrium experiments at protein concentrations greater than 0.02 mg/ml.

In the XL-I interference optical system, a beam of monochromatic light is directed through a sector containing solute and a second beam passes through an adjacent sector containing solvent to create an interference or fringe pattern, consisting of a series of bright and dark lines.  As the sample sediments, changes in the protein concentration at the boundary region lead to differences in the refractive index that result in a displacement of the fringe pattern.  Interference optics can be used for sedimentation velocity and sedimentation equilibrium experiments at protein concentrations greater than 0.1 mg/ml.  This system is particularly useful for analysis of high molecular weight samples or complex mixtures in sedimentation velocity experiments, because scans can be collected more rapidly and at a higher frequency than those using absorbance optics.  It is also useful for analysis of associating systems, as fringe displacement is proportional to sample concentration over a wide range.  Furthermore, because virtually all materials change a solvents refractive index, the interferometer can detect samples containing no natural chromophores.

Sedimentation velocity

A relatively high rotor speed is used to cause rapid sedimentation of solute towards the bottom of the sample cell. This causes depletion of solute from the meniscus creating a boundary between the depleted region and the uniform concentration of solute. The rate of movement of this boundary gives the sedimentation coefficient s. This parameter is dependent both on the mass of the sedimenting species and the frictional coefficient, which in turn is a measure of its effective size. Techniques which provide an estimate of the frictional ratio, such as gel filtration chromatography or light scattering can be combined with sedimentation experiments to enable calculation of the native molecular mass.

A lower rotor speed is used than is required for complete sedimentation of the sample. Sedimentation is opposed by diffusion such that a gradient of protein concentration is set up across the cell. Analysis of the concentration gradient at equilibrium enables calculation of the native molecular mass of the sample independent of its shape. Equilibrium experiments can be used for determining the native molecular mass of samples and for analysing self-associating systems.

The sample must be as pure as possible and free from contaminating proteases.  Prior to setting up each experiment samples should be dialysed against buffer, and the dialysate used in the reference channel.  If absorbance optics is to be used, buffer should be selected which does not absorb significantly in the chosen wavelength range.  Nonideal behaviour, which can hinder analysis of complex systems, can be minimised by including 0.1 M NaCl or KCl in the buffer.

For a single experiment, 400 microlitres of sample and 425 microlitres of sample buffer are loaded into the sample and reference channels of each sample cell. When using absorbance optics, a wavelength is chosen to give an initial absorbance of between 0.5 - 1.0. The lowest concentration of protein that can be used is ~ 0.1 mg/ml for most samples. Rotor speeds of up to 55,000 rpm enable effective sedimentation of proteins greater than 5,000 Da. Typical experiments take up to 6 hours to complete. Samples in three different sample cells can be centrifuged simultaneously. Analysis software is available for calculation of the sedimentation coefficients of single species and more complex systems where more than one species is present.

A total volume of 200 microlitres of sample at a minimum concentration of ~ 0.02 mg/ml (0.1 mg/ml for interference optics) is sufficient for setting up each sample cell. Individual sections within each cell are loaded with three different concentrations of sample (110 microlitres) at two-fold dilutions since these give overlapping data sets at equilibrium. The corresponding reference channels are loaded with buffer (125 microlitres or 115 microlitres for interference optics). When using absorbance optics, a wavelength is chosen to give an initial absorbance of 0.5 for the most concentrated sample. Three cells can be run simultaneously. Samples can take several days to reach equilibrium. Analysis software is provided for determination of the native molecular mass of single species and for analysis of self-associating systems with up to four components.

Training can be provided for setting up centrifugation runs and for use of the analysis software. Only one training session can be given for each research group wishing to use the centrifuge. New members of a group must be trained internally and group leaders are expected to ensure that training is passed along in a responsible way when group members leave. The training sessions are designed to enable users to set up experiments, to prepare data for analysis and to run the analysis programs. Interpretation of the data is left to individual users. Some references which provide an introduction to analytical ultracentrifugation are given below.

Any publications that include data generated using this facility should acknowledge the funding provided by the Wellcome Trust and BBSRC.  In addition, details of the publication should be sent to Russell Wallis.

          All bookings must be through

                    Russell Wallis
                    Glycobiology Institute
                    Department of Biochemistry
                    Telephone       01865 275762
                    E-mail          rwallis@glycob.ox.ac.uk

This is the only way to sign up for training or for use of the instrument.

Introduction to Analytical Ultracentrifugation Order No: 361847
Self-associating systems in the Analytical Ultracentrifuge Order No: 362784

Hansen, J.C., Lebowitz, J. and Demeler, B. (1994). Analytical ultracentrifugation of complex macromolecular systems. Biochemistry33, 13155-13163.
Hensley, P. (1996). Defining the structure and stability of macromolecular assemblies in solution: the re-emergence of analytical ultracentrifugation as a practical tool. Structure4, 367-373.

Extensive reference lists, further information about the analytical ultracentrifuge and analysis software can be obtained from:

http://biochem.uthscsa.edu/auc
http://www.beckmancoulter.com/Beckman/biorsrch/prodinfo/xla/xlaprod.asp