Band-centrifugation of macromolecules and viruses in self-generating density gradients

Abstract

This communication presents a new method of carrying out sedimentation velocity experiments. A thin lamella of a solution of macromolecules is layered onto a denser miscible liquid in a rotating ultracentrifuge cell. The macromolecules then sediment through the liquid in a narrow concentration distribution, or band, which is observed photographically as a function of time. The density gradients necessary to stabilize the system against convection are generated during the experiment by the diffusion of small molecules between the lamella and the bulk solution, and in some cases by the sedimentation of the small molecules in the bulk solution. The inhomogeneities in the solvent are usually small enough to have no observable effect on the motion of the macromolecules. Sedimentation coefficients of macromolecules may be evaluated from the motion of bands. Diffusion, hydrodynamic interactions, and chemical reactions affect the shapes of bands.