Isolation of centrosomes from cultured cells.

The centrosome is a unique organelle that nucleates and organizes the interphase microtubule array and facilitates bipolar spindle assembly during mitosis. Isolated centrosomes are ideal starting materials for biochemical and structural studies as well as for functional assays to study microtubule-dependent processes. In fact, they are hitherto the only system to study radial microtubule arrays in vitro. This chapter provides a practical method and the rationale of isolating centrosomes from adherent cultured cells by density-gradient centrifugation. It further describes how to evaluate the activity and function of centrosomes by microtubule nucleation and immunofluorescence assays and how to measure microtubule dynamics nucleated from isolated centrosomes in Xenopus egg extracts.