1. Analytical Ultracentrifugation

3. Analytical Ultracentrifugation – Applications
Determine sample purity
Characterize assembly and disassembly mechanisms of biomolecular complexes
Determine subunit stoichiometries
Detect and characterize macromolecular conformational changes
Measure equilibrium constants and thermodynamic parameters for self- and hetero-associating systems  characterize the solution-state behavior of macromolecules under various conditions

4. Analytical Ultracentrifugation – Design
analytical ultracentrifuge = preparative ultracentrifuge + optical detection system  measure sample concentration inside the centrifuge cell during or after sedimentation
centrifugation parameters and data acquisition under computer control  experiments lasting many days performed with minimal operator intervention

6. Analytical Ultracentrifugation – Design

7. Analytical Ultracentrifugation – Design: Optical systems
Absorbance optical system:  measurement of sample concentration at wavelengths from 200 to 800 nm  detection of macromolecules containing strong chromophores
Rayleigh interference optical system:  measurement of sample concentration based on refractive index changes  analyze macromolecules lacking intense chromophores (eg, polysaccharides) and samples that contain strongly absorbing buffer components (eg, ATP/GTP, DTToxidized)

8. Cindy Horwedel

9. 1-Analytical Ultracentrifugation – Sedimentation velocity experiments

10. 1-Analytical Ultracentrifugation – Sedimentation velocity experiments
Meniscus of solvent
Sedimentation front
Meniscus of solution
Plateau conc.
Cell radius
Concentration of solute
Modified from

11. Experimentally, the data is gathered at high rotor speeds where the sedimentation transport dominates the diffusion.
One measures a molecule's rate of transport from the top to the bottom of the cell.
A sedimentation velocity experiment requires approximately 500 µg of sample protein in a volume of 1 ml.

12. 1-Analytical Ultracentrifugation – Sedimentation velocity experiments
Hydrodynamic information
Experimentally determined parameters:
Sedimentation coefficient s
Diffusion constant D or friction factor f
Molecular mass M
Estimation of the molecule’s shape in solution

13. 2-Sedimentation Equilibrium provides thermodynamic information
Experimentally, the data are collected at slower rotor speeds so that the sedimentation and diffusion forces can balance.
At sedimentation equilibrium, these forces are equal in magnitude, but opposite in direction, and the molecule is exponentially distributed across the cell.
There is no longer any net transport of molecules in the system. All shape factors are thus cancelled out, yielding the molecular weight.
A typical sedimentation equilibrium experiment requires 300 µl of sample at an OD of 0.8 at the wavelength of interest (usually 280 nm). This is generally on the order of µg of total protein, but varies greatly depending on your protein's molar extinction coefficient.

16. Analytical Ultracentrifugation – Examples of Applications
Sedimentation velocity
Biomolecular Shape
Biomolecular Conformational Changes
Assembly and Disassembly of Biomolecular Complexes
Molecular Mass and Subunit Stoichiometry
Equilibrium Constants for Self-Associating Systems
Sedimentation equilibrium
Equilibrium Constants for Molecules