Centromere and Kinetochore Assembly in Egg Extract.

During cell division, chromosomes must be equally segregated to daughter cells. Centromeres, the primary interaction site between chromosomes and microtubules, mediate faithful chromosome segregation during mitosis. Functional studies of centromere proteins in cells have proven difficult, as mutation or deletion of most centromeric proteins often results in cell lethality. In this protocol, sperm chromatin or reconstituted chromatin arrays, together with egg extracts, are used to overcome these limitations and study centromere and kinetochore assembly in vitro. egg extract is a powerful tool, as it can be readily cycled in vitro by addition of calcium and easily modified biochemically. Coupled with the addition of customizable reconstituted chromatin arrays or sperm chromatin, egg extract provides distinct advantages over cell-based approaches in which similar experiments would not be feasible. Following incubation in egg extract, reconstituted centromeric chromatin arrays and sperm chromatin specifically assemble core centromere and kinetochore components that can be analyzed via immunofluorescence.