Analytical Ultracentrifugation (AUC)

Study the size and shape of macromolecules in solution to facilitate structural determination of large proteins

 
Christine Ebel Christine Ebel email hidden
Robert Gilbert Robert Gilbert email hidden

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Analytical Ultracentrifugation (AUC) Details

User Guide

The quantity of material required for AUC experiments is typically in the order of a few hundred micrograms and volumes are in the range 100 – 400 µl. The purity of the sample should be greater than 95%. Depending on the optical detection system used, loading concentrations are in the range 0.05 - 2 mg/ml or 0.05 - 1.5 OD. For the study of interactions, up to three protein components can be mixed. Interacting components under study may have sizes ranging from peptides to very large macromolecular complexes like ribosome particles. In general, affinities in the range of 104 to 108 M-1 can be determined and kinetic dissociation rate constants in the order of 10-5 – 10-2 sec-1. Samples should be equilibrated into the experimental buffer which serves as a reference (gel filtration or dialysis should be used to match the reference buffer). Most of the commonly used buffer components are compatible with AUC experiments. Sedimentation velocity experiments should take 3-6 hours and sedimentation equilibrium experiments 2-5 days.

Technical Specifications

The analytical ultracentrifuge may include three optical detection systems:

  1. an absorbance system with a wavelength range from 190 to 800 nm to detect macromolecules through their extinction properties,
  2. a Rayleigh interferometer to detect macromolecules through refractive index changes and
  3. a fluorescence optical system with excitation at 488 nm to detect labeled compounds.

The analytical rotor may hold 4 to 8 analytical cells. Each analytical cell contains a centerpiece with 2 or 6 channels sealed between 2 transparent windows in a housing cell. The analytical ultracentrifuge is controlled by the XL-A/XL-I user interface software which allows the entry of analytical run settings and the ability to display and save data to disk.