155Chapter 10
Measurement of Caspase Activation in Mammalian
Cell Cultures
Magnus Olsson and Boris Zhivotovsky
Abstract
The majority of caspases, cysteine-dependent aspartate-directed proteases, being in their activated state are
involved in regulation of apoptosis by cleaving protein substrates harboring specifi c target motifs. Basically
all biochemical and morphological changes in an apoptotic cell, including cell shrinkage, chromatin con-
densation, DNA fragmentation, and plasma membrane blebbing, are consequence of caspase-mediated
proteolysis. Thus, uncovering activities of unique caspases are key determinants of the apoptotic process.
This chapter describes a set of experimental protocols available for characterization, quantifi cation and
inhibition of caspase activities in mammalian cell cultures, including immunoblotting, usage of synthetic
substrates, fl ow cytometry, and microscopic techniques.
Key words Apoptosis , Caspase , Caspase substrate , SDS-PAGE , Immunocytochemistry , FACSAbbreviations
BSA Bovine serum albumin
FACS Fluorescence activated cell sorting
pAb Polyclonal antibody
SDS Sodium dodecyl sulfate
Tris Tris(hydroxymethyl)aminomethane, mAb, monoclonal antibody
1 Introduction
In a simplifi ed scheme, an individual caspase is synthesized as a
zymogen, and proteolytical processing of the precursor is then
required for the formation of a tetrameric active enzyme, com-
posed of two repeating heterotypic subunits. The fi rst process-
ing event in a procaspase is separation of the large (p17) and
small (p12) subunits, which is followed by deletion of the N-
terminal pro-domain to form the catalytically mature protease [ 1 ].
Caspase zymogens are themselves substrates for caspases and1.1 Immunoblot
Analysis of
Procaspase
Processing
Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_10, © Springer Science+Business Media New York 2014