Analytical Ultra Centrifuge

The equipment at the Research Complex at Harwell includes the Beckman Coulter ProteomeLab XL-I Protein Characterization System. This Analytical Ultracentrifuge has two integrated detection systems, Rayleigh Interference and Scanning UV/VISIBLE optics, and is designed to detect, measure, record and analyze the movement of molecules in solution under a centrifugal field. The system is controlled by WindowsXP-user interface software that allows users to set parameters, initiate a run and collect data. Data Analysis software calculates hydrodynamic and thermodynamic properties of macromolecules including sedimentation and diffusion coefficients, association constants (and more) informing sample heterogeneity and stoichiometry, and the molecular conformation of interacting and self-associating systems. The AN-60 Ti rotor can spin up to 60,000rpm, generating up to 290,000 x g.

Analytical System Specifications

Equipped with integrated Rayleigh Interference and Scanning UV/VISIBLE detection systems.

Rayleigh Interference:

Analyses macromolecules lacking intense chromophores (eg: polysaccharides) and samples containing strongly absorbing buffer components (eg: ATP/GTP, DTToxidized). It measures sample concentrations based on refractive index changes and can characterize very concentrated samples.

  • Wavelength 660nm
  • CCD Camera Resolution 2048 x 96 pixels
  • Laser 30mW Diode
  • Scan Rate approximately every 5 seconds
  • Interferometer precision approximately +/- 0.003 fringe

Scanning UV/VISIBLE detection system

The Scanning UV/Vis detection system allows selective detection of all molecules that show absorption at a specific wavelength, and allows characterisation of separated particle populations according to their absorption profile.

  • Operates in wavelength or radial scanning mode
  • Detection wavelength ranges from 190 to 800nm
  • 0 to 3 Å photometric display range
  • Optical pathlength is 1.2cm or 0.3cm
  • Minimum radial increment 0.001cm
  • Minimum wavelength increment 1nm
  • Maximum of 1000 data points per scan
  • Approximately 1 absorption reading every 20 milliseconds
  • 1 to 100 replicates per data point


Comprehensive Data Analysis Software Package

  • Scripts for equilibrium and velocity analyses
  • Defined graphical and numerical output, easily customized
  • Calculates parameters include equilibrium association constants (Ka), solution and molecular weights, sedimentation and diffusion coefficients

Sample concentrations and volumes

Protein and nucleic acid concentrations from approximately 10µl/ml to 5mg/ml
Volumes:

~420µl for sedimentation velocity experiments (optical pathlength 1.2cm)
~110µl for standard sedimentation equilibrium (optical pathlength 1.2cm)

Uses

There are two 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 a series of 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 biomolecular shape and conformational shape changes; molecular mass and sub-unit stoichiometry; assembly and disassembly of biomolecular 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 hetero-associating systems.
SV and SE provide complementary information and are often both applied to the same problem.