Analytical Ultracentrifugation Experiments

Sedimentation velocity (SV)

SV is a time-dependent technique in which particles of interest sediment throughout the duration of the experiment, ultimately pelleting to the bottom of the cell.

In SV experiments, an initially uniform solution is placed in the cell, and a sufficiently high angular velocity is used to cause relatively rapid sedimentation of solute toward the cell bottom. This produces a depletion of solute near the meniscus, and the formation of a sharp boundary between the depleted region and the uniform concentration of sedimenting solute. 

Though the velocity of individual particles cannot be resolved, the rate of movement of this boundary can be measured. This leads to the determination of the sedimentation coefficient, s, which depends directly on the mass of the particles and, inversely, on the frictional coefficient, which is in turn a measure of effective shape. This can be represented by the equation:

Sedimentation Velocity Image AUC Formula

SV is generally considered the primary method of choice for most current applications of AUC, including the study of protein self- and hetero-association, the study of membrane proteins, and applications in biotechnology.1

Sedimentation equilibrium (SE)

This is a time-independent AUC technique in which the particle of interest reaches an equilibrium state in the centrifuge cell and does not pellet.

In SE experiments, a small volume of an initially uniform solution is centrifuged at a lower angular velocity than is required for an SV experiment. As solute begins to sediment toward the cell bottom and the concentration at the bottom increases, the diffusion process opposes the sedimentation process. 

Sedimentation Equilibrium Image AUC Formula

Ultimately, the two opposing processes approach equilibrium, and the concentration of the solute increases exponentially toward the cell bottom. At equilibrium, the resulting solute distribution is invariant with time. Measurement of the concentration at different points can determine the molar weight of the sedimenting solute (numerically equal to the molecular weight). Read more

 

TESTIMONIAL

“Due to the large size and conformational heterogeneity of our samples, sedimentation velocity experiments in the analytical ultracentrifuge have allowed us to make many seminal findings about chromatin condensation mechanisms that would not have been possible with any other technique.”

Jeffrey C. Hansen, Ph.D. 
Colorado State University 

 

References

Zhao H, Brautigam CA, Ghirlando R, et al. Current Methods in Sedimentation Velocity and Sedimentation
Equilibrium Analytical Ultracentrifugation. Curr Protoc Protein Sci 2013;20:2-52.