Preparation of cytosolic extracts and activation of caspases by cytochrome c.
It can be useful to explore the caspase activation process in an in vitro setting.  In this protocol, cytosolic extracts prepared from cell culture are incubated with cytochrome c and adenosine triphosphate (dATP), leading to the oligomerization of apoptotic protease activating factor-1 (APAF-1) and the formation of the apoptosome.  The apoptosome serves as an activation platform for caspase-9, which binds to the apoptosome through heterodimeric caspase recruitment domain (CARD) interactions and then dimerizes.  This leads to cleavage of the executioner, caspase-3.  These extracts contain highly active caspases that can be analyzed using a variety of biochemical assays.