Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021:2255:1-12.
doi: 10.1007/978-1-0716-1162-3_1.

Determination of Caspase Activation by Western Blot

Hussein Chehade  1 , Alexandra Fox  1 , Gil G Mor  1 , Ayesha B Alvero  2
Affiliations

Determination of Caspase Activation by Western Blot

Hussein Chehade et al. Methods Mol Biol. 2021.

Abstract

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.

Keywords: Apoptosis; Caspases; Cleavage; Extrinsic Pathway; Proteins; Western Blot.

PubMed Disclaimer

References

    1. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516. https://doi.org/10.1080/01926230701320337 - DOI - PubMed - PMC
    1. Taylor RC, Cullen SP, Martin SJ (2008) Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 9(3):231–241. https://doi.org/10.1038/nrm2312 - DOI - PubMed
    1. Degterev A, Boyce M, Yuan J (2003) A decade of caspases. Oncogene 22(53):8543–8567. https://doi.org/10.1038/sj.onc.1207107 - DOI - PubMed
    1. Nair P, Lu M, Petersen S, Ashkenazi A (2014) Apoptosis initiation through the cell-extrinsic pathway. Methods Enzymol 544:99–128. https://doi.org/10.1016/B978-0-12-417158-9.00005-4 - DOI - PubMed
    1. Kallenberger SM, Beaudouin J, Claus J, Fischer C, Sorger PK, Legewie S, Eils R (2014) Intra- and interdimeric caspase-8 self-cleavage controls strength and timing of CD95-induced apoptosis. Sci Signal 7(316):ra23. https://doi.org/10.1126/scisignal.2004738 - DOI - PubMed - PMC

LinkOut - more resources