Preparation of Cytosolic Extracts and Activation of Caspases by Cytochrome c

Gavin P. McStay; Douglas R. Green

doi:10.1101/pdb.prot080275

Preparation of Cytosolic Extracts and Activation of Caspases by Cytochrome c

Gavin P. McStay1,3 and
Douglas R. Green2

¹Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568;
²Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Abstract

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.