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Chapter category: Apoptosis

In Situ Activation of Caspases Revealed by Affinity Labeling Their Enzymatic Sites

This chapter appears in the following book:

Caspases: Their Role in Cell Death and Cell Survival

Edited by: Marek Los and Henning Walczak
ISBN: 0-306-47441-7
» Get more information about this book at landesbioscience.com «

Chapter authors:
Jerzy Grabarek and Zbigniew Darzynkiewicz


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Activation of caspases is the key event of apoptosis as its initiates irreversible steps of the cell demise.1-9 Several methods, therefore, have been developed to monitor this event. Most frequently the caspases involvement is probed indirectly, by testing whether their specific inhibitors, when administered together with the inducer of apoptosis, can prevent particular apoptotic episodes. Also indirectly, caspases activation can be revealed by the presence of the specific cleavage products that can be identified electrophoretically by a characteristic change in molecular weight upon the cleavage, confirmed immunochemically on Western blots. Antibodies also are available that detect the specific cleavage products such as a 89 kD fragment of poly(ADP-ribose) polymerase-1 (PARP-1).10 The latter approach was adapted to cytometry, and has been utilized to correlate caspases activation with the cell cycle position9 or collapse of the mitochondrial transmembrane electrochemical potential.11

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