Analytical Ultracentrifuge (AUC)

RCaH has two Beckman Coulter analytical ultracentrifuges (AUCs): an Optima AUC and a ProteomeLab XL-I. Each instrument has two integrated detection systems: Rayleigh Interference and Scanning UV/Visible optics, which are designed to detect, measure, record and analyse the movement of molecules in solution under a centrifugal field. The Optima AUC, one of only two currently in the UK, offers faster data collection, at higher resolution, and is particularly suited to multi-wavelength experiments.

There are two main types of analytical ultracentrifuge experiment: sedimentation velocity (SV) and sedimentation equilibrium (SE).

SV is a hydrodynamic technique, sensitive to the mass and shape of the macromolecule. A moving boundary is formed on application of a strong centrifugal force and scans are recorded at regular intervals, which determines the rate of movement and the broadening of the boundary as a function of time. SV can be used to determine molecular shape and conformational shape changes, molecular mass and sub-unit stoichiometry, assembly and disassembly of molecular complexes. It is ideally suited for the analysis of heterogeneous samples.

SE is a thermodynamic technique and is sensitive to the mass but not the shape of the macromolecule. Experiments are performed at lower speeds and measure the equilibrium concentration distribution of macromolecules formed when sedimentation is balanced by diffusion. SE can be used to determine absolute molecular mass of the complexes present in solution, and equilibrium constants for self-associating and hetero-associating systems.

SV and SE provide complementary information and are often both applied to the same problem.

SV and SE can be combined in a gravity sweep experiment, which facilitates analysis of samples over a very large range of sedimentation coefficients. This is particularly suitable for characterising nanoparticles in solution.