Determination of Caspase Activation by Western Blot.

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.