Chapter 5 Hydrodynamic Analysis of Human Kinetochore Complexes During Mitosis

Hydrodynamic analysis is a powerful tool to dissect the molecular architecture of macromolecular protein assemblies. These techniques have been successfully used in yeast systems but are also well suited to the analysis of protein complexes from human cells. Furthermore, the combination of hydrodynamic analysis with siRNA mediated protein depletion provides an excellent system to probe the composition of protein complexes isolated from human cells. In this chapter we describe the use of these approaches in the analysis of macromolecular protein complexes during mitosis in human cells, using the kinetochore as an example. Key words: Size-exclusion chromatography, sedimentation equilibrium, glycerol gradient, Svedberg coefficient, Stokes radius, kinetochore, mitosis