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Analytical ultracentrifugation (AUC)

AUC with fluorescence detection system

Analytical ultracentrifugation is a powerful technique of characterizing molecules in solution. The information comes from first-principle hydrodynamic and thermodynamic measurements. The concentration dependence of the molecular weight can provide the same thermodynamic information as light scattering. The rate of movement of molecules provides hydrodynamic size information.

  • CAMIS developed the Fluorescence Detection System (FDS) that is now marketed by Aviv Biomedical, Inc. for installation into the Beckman Coulter ProteomeLab XL-A/XL-I or Proteome systems.
  • Specialty cells and accessories, such as the cell washer for double sector cells, developed at CAMIS, are now available exclusively at Spin Analytical, Inc.
  • Advancement of components and software for the AUC and FDS continues in conjunction with Spin Analytical and academic labs. Such projects include a system for automating the interpretation of AUC data.

AUC at CAMIS

cell parts

CAMIS has two Beckman-Coulter XL-A/XL-I instruments equipped with all 3 detection systems – absorbance, interference and fluorescence. The centrifuges are available for both collaborative and fee-for-service use. For more information please contact us or visit services.

Key features of AUC

  • Detection over a wide concentration range – nM to µM
  • Analysis over a wide size range – nm to µm
  • Small volumes (10-480 µL depending on experiment)
  • Does not require a standard
  • Absorbance, interference, and fluorescence detection are available
  • Sample can be recovered

AUC can provide characterization of macromolecules for:

bullet Molecular weight bullet Size
bullet Heterogeneity bullet Shape
bullet Stoichiometry bullet Non-ideality
bullet Association

Choosing an Optical System:

Absorbance
  • Has selectivity 190 – 800 nm
  • Good sensitivity - minimum 0.1 OD
  • Use if cannot dialyze the sample
  • Easy to use
  • Good precision
  • Good resolution
Interference
  • Use if buffer absorbs
  • Use if sample does not absorb
  • Excellent precision
  • Use if extinction coefficient varies
  • Can use short column centerpieces for small amount of sample
  • Good sensitivity – 50 µg/ml
  • No selectivity
  • Requires sample dialysis
Both Absorbance and Interference
  • Determine extinction coefficient
  • Test for sample purity
  • Extend concentration range
Fluorescence
  • Excellent sensivity – 500 pM
  • Excellent selectivity – can run sample in complex background solution (cytosol, serum)
  • But requires label
  • Analysis of tight associations (Kd < 10-9 M).
  • Fair precision
  • Good resolution

Sedimentation Velocity vs Equilibrium

There are two basic types of sedimentation experiments. In sedimentation velocity experiments, the speed of the run is such as to sediment the molecule relatively quickly (hours). The rate of movement of the boundary is used to determine the sedimentation coefficient (s). From sedimentation coefficient, the purity of the sample, the number of species, hetero- or self-association, hydrodynamic size and shape can be determined.

In sedimentation equilibrium experiments the speed and length of the run (hours to days) is such that the molecules movement in the gravitational field is balanced with diffusion. The analysis of the concentration profile provides information on the molar mass, stoichiometry, association constants and non-ideality.