Study the size and shape of macromolecules in solution to facilitate structural determination of large proteins
Instruct has 4 centres offering Analytical Ultracentrifugation (AUC) across Europe. Navigate the map and click on the pins to discover centres near you.
Analytical ultracentrifugation (AUC) is a powerful technique for the characterisation of macromolecules and macromolecular self- and hetero-association processes in solution. Two types of complementary experiments, sedimentation velocity and sedimentation equilibrium, can be performed in an analytical ultracentrifuge which is a high-speed centrifuge equipped with an optical detection system. The observation of macromolecules or macromolecular complexes sedimentation gives access to their hydrodynamic and thermodynamic properties, including their size, shape, molar mass, degree of heterogeneity, oligomeric state, stoichiometry, and binding constants.
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.