Analytical Ultracentrifugation
Analytical ultracentrifugation is a direct biophysical analysis method yielding information about the mass, structure and interactions of purified proteins and other biomolecules in dilute solutions. The technique studies their hydrodynamic behaviour when subjected to a centrifugal field
Biological areas of interest amenable to analytical ultracentrifugation study include:
• Protein mass, shape and stoichiometry
• Protein-protein/lipid/nucleic acid interactions
• Membrane proteins and their complexes
• Glycosylated proteins and their complexes
Analytical centrifugation offers two highly complementary methodologies: sedimentation velocity and sedimentation equilibrium.
Sedimentation velocity is an experiment which measures how fast macromolecules move in response to centrifugal force. Measuring changes in sedimentation boundary movement gives us information about the mass and shape of macromolecules.
Sedimentation velocity experiments are performed at high speeds of 30,000 – 50,000 rpm and can address sample-related questions concerning:
• Homogeneity / heterogeneity in protein mass & conformation
• Determining sedimentation coefficients of solutes
• Detecting the presence of aggregates and their size distribution
• Determination of overall shape/asymmetry of macromolecules
• Define molecular mass and stoichiometry
Sedimentation equilibrium is based on balancing the opposing forces on a particle of centrifugal sedimentation and diffusion out of the resulting concentrated solute phase, ending in a gradient which remains constant for an indefinite period of time. Such experiments are generally performed at lower rotation speeds and so take several days to complete.
The technique permits determination of:
• Molecular mass and subunit stoichiometry
• Molecular dissociation constants (KD)
• Second virial coefficient (B) – the measure of non-ideality of the solute (due to protein shape or charge)
At Pinnacle our complete AUC service includes the following:
• Formulation of the initial model
• Building any prior knowledge into the model
• Fitting all start-to-end experimental data to the model
• Treating all sedimentation experiments globally
• Reconciling sample sedimentation / hydrodynamic analysis data with other biophysical analysis such as x-ray/neutron small angle scattering, dynamic light scattering, calorimetry, etc.
Equipment
The analytical ultracentrifuge is a high speed instrument equipped with an optical system for observing the sedimentation process of biomolecules in real time.
Our ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) has the following specifications:
• UV/Vis wavelength range 190-800 nm
• Radial scans at three wavelengths possible during one experiment
• Simultaneous interference scanning can be performed
• Range of mass determination from 1- 5000 kDa (i.e. from peptides to viruses)
• Concentration range for absorbance scanning of 0.05-2 mg/ml & for interference scanning of 0.025-30 mg/ml
• Dissociation constant measurement range of 10-3-10-8 M
We have an 8-hole rotor (AN50Ti), 2-sector and 6-sector cells with a choice of quartz or sapphire windows. The rotor can accommodate up to 7 samples (of biomolecule measured against its buffer) for sedimentation velocity runs and up to 21 samples for sedimentation equilibrium runs.
The absorbance system consists of a double-beam spectrophotometer coupled with a slit which scans from the top to the bottom of the cell. The advantages of this detection are UV/Vis selectivity and tolerance of an imperfectly matched reference buffer. Disadvantages include long scanning times (90 sec to scan one cell) and comparatively noisy signal.
The Rayleigh interference system detects solute distribution by differences in refractive index between sample and buffer solutions. Interference detection has advantages in terms of uv-transparent compounds, detection speed (10 sec to scan one cell) and better signal-to-noise ratio. It is essential that sample and buffer have equal chemical potential (i.e. should be thoroughly dialysed) and column heights in sample and buffer sectors of the cell should be of the same length. These two requirements are important for this type of detection.
Interference and absorbance scans can be performed simultaneously if required.
Sample Preparation
All samples intending to undergo analytical ultracentrifugation experiments should be thoroughly dialysed against buffer (gel-filtration buffer is also suitable). The buffer should contain at least 100 mM salt and the best choice of initial experimental buffer would be either phosphate or Tris-buffer containing about 150 mM NaCl.
Usually we ask for 2 mg of protein (the desired volume for any stock solution is about 1 ml) - this should provide enough material to carry out both sedimentation velocity and sedimentation equilibrium experiments. Analytical ultracentrifugation is a non-destructive method and you can get your sample back at the end if you wish. Please also supply 10-15 ml of dialysis/gel-filtration buffer.
If you have not got enough protein, cannot use the recommended buffer or cannot dialyse the sample for some reason please consult with Alex Solovyova [Tel. X.3504].
Sample Submission (Additional Information)
We recommend that you discuss any proposed analytical ultracentrifugation studies with us well in advance. Details about your sample/system are required before setting-up an AUC run in order to ensure the best possible conditions for that run can be met.
It is therefore important that prior to analysis you complete our additional sample information form <click here> and bring it to the unit together with your sample.
With regard to charges / payment, on-campus sample orders must be placed using our web forms <here>.
Please remember, the more information you can supply about your sample, the better the analysis that can be performed.