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Analytical Ultracentrifugation
(AUC)

The Beckman ProteomeLab XL-1 measures concentration distributions of a sample in solution in a centrifugal field.  The application of centrifugal force causes a redistribution of the macromolecules and the formation of a concentration boundry that moves from the meniscus to the bottom of the cell over time.  A significant strength of technique is that the properties of native proteins are studied in solution -requiring no label, chemical modification or surface interaction.

The XL-1 is equipped with two optical detection systems.  The first is capable of reading the absorbance of a sample using a dual-beam UV/VIS spectrophotometer with monochromator.  The second detection system uses a laser interferometer that records the refractive index gradients.  The choice of the optical detection system depends on the sensitivity needed for the experiment, the concentration range to be used, the extinction properties of the system and the buffer properties.

Two methods used for running the analytical ultracentrifuge are sedimentation velocity and sedimentation equilibrium.  In sedimentation velocity, high rotor speed sediments macromolecules to the bottom of the cell.  The rate of sedimentation is dependent on the size and the shape of the protein.  Sedimentation velocity experiments generally run for 6 - 12 hours.   Sedimentation equilibrium experiments are run at lower rotor speeds where the process of sedimentation is balanced by diffusion.  When no change in the concentration distribution is detectable, sedimentation equilibrium is achieved.  The time required to establish sedimentation equilibrium is generally 1 -2 days with the entire experiment typically taking between 3 and 5 days.

Applications

  • Determine the molar mass of proteins and complexes
  • Assess the purity of a sample
  • Determine the number of species in a sample
  • Determine the stoichiometry of complexes
  • Analysis of self- and hetero-association
  • Determine binding constants (10-3– 10-8 M)
  • Characterizes synthetic polymers, biological macromolecules, viral particles, carbohydrates and nanoparticles

Specifications

  • Temperature range: 0oC to 40oC
  • Rotor speed: 1 - 60K
  • Equipped with both absorbance and interference optics
  • Absorbance optical range: 190 - 800 nm; linear to 1.5 OD
  • Interference opticaly system reads at 660 nm
  • An-50 Ti rotor: 8 hole (7 sample cells plus counterbalance); max speed 50K
  • An-60 Ti rotor: 4 hole (3 sample cells plus counterbalance); max speed 60K
  • Sample cells:
    • 3-12mm double sector Epon charcoal centerpieces
    • 3-12mm double sector aluminium centerpieces
    • 3-12mm equilibrium 6-channel Epon charcoal centerpieces with external fill
  • Quartz and sapphire windows

Buffer Requirements

  • Ionic strength of the buffer should be >10 mM (100 - 200 mM is recommended) to limit electrostatic repulsion
  • Any buffer component that raises the density of the buffer should be avoided as this may cause concentration gradients at high rotor speeds
  • When using ABSORBANCE optics, the buffer should be transparent at the aquisition wavelength (<0.2 OD).  HEPES and Tris buffers absorb in the far UV but low concentrations are tolerable (~10 mM).
  • DTT should be avoided as its sepctroscopic properties change over time.  TCEP is a more stable alternative.
  • Nucleotides can be used but at a concentration >50 mM cannot be used with absorbance optics (use interference optics instead)

Sample Requirements

  • The sample does not have to be spectroscopically active
  • Sample and buffer used in the reference cell MUST be an EXACT chemical match.  Achieved by exhaustive dialysis (use last dialysis buffer as reference) or gel filtration.  The instrument can detect minor buffer mismatches which will complicate analysis.
  • Sample concentration: 0.1 - 1.5 OD 
  • Sample volume:
    • 2-sector centerpiece: 450 ml sample
    • 6-sector centerpiece: 150 ml sample
  • Reference buffer volume: 10 ml
  • Sample stability: >5 hours for sedimentation velocity and 2 - 5 days for sedimentation equilibrium

Supplies You Should Bring for Your Experiment

  • Buffer and sample as outlined in the sample requirements section
  • Housing plugs - order from Beckman Coulter, part # 327022 (~$50/pkg of 100).  You will need 4 non-reusable plugs per sample.
  • Window gaskets if you plan on using the aluminium centerpieces - you will need 2 gaskets per sample and the gaskets are non-reusable
    • order from Beckman Coulter, part # 330446, pkg of 10 (~$60)
  • CD or USB drive for saving your data (only if you want the raw data files)

Training, Assistance or Instrument Problems

If you wish to use the analytical ultracentrifuge, you must find a qualified user to train you on the operation of the instrument.

Manual and Articles

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