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Updated: Apr 27, 2026
Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Published on: January 20, 2023
Detection of caspase activity using antibody-based techniques
Gavin P McStay1, Douglas R Green2
1Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568;
Abstract:
A number of antibodies have been generated that recognize caspases from mammalian model organisms. These include antibodies that recognize specific caspase pro-forms and others that bind caspase cleavage fragments. These antibodies are excellent reagents for identifying which executioner caspases have been activated following application or induction of a specific apoptotic stimulus. This approach is more difficult to use with initiator caspases, however, because cleavage does not necessarily correlate with caspase activation. In this protocol, cultured cells are treated with a proapoptotic stimulus, and then protein lysates are prepared from the treated cells. The proteins are then separated by gel electrophoresis and transferred to a suitable membrane. The fragment-specific antibodies that recognize executioner caspases are used in a western analysis to determine the extent of activation and to aid in identifying which caspases have been activated.
Insights
Antibodies targeting caspase cleavage fragments can identify activated executioner caspases in mammalian cells. This western blot protocol aids in determining caspase activation following apoptotic stimuli.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Caspases are key proteases in apoptosis, with specific antibodies available for mammalian models.
- Antibodies can target either caspase pro-forms or their cleavage fragments.
- Detecting activated executioner caspases is crucial for understanding apoptosis.
Purpose of the Study:
- To present a protocol for identifying activated executioner caspases using fragment-specific antibodies.
- To enable the determination of caspase activation extent and identity following apoptotic stimuli.
Main Methods:
- Treatment of cultured cells with a proapoptotic stimulus.
- Preparation of protein lysates from treated cells.
- Western blot analysis using fragment-specific antibodies to detect cleaved executioner caspases.
Main Results:
- Fragment-specific antibodies effectively identify activated executioner caspases.
- Western blot analysis quantifies the extent of caspase activation.
- This method aids in pinpointing which specific caspases are activated.
Conclusions:
- Antibodies recognizing caspase cleavage fragments are valuable tools for apoptosis research.
- The described western blot protocol provides a reliable method for assessing executioner caspase activation.
- This approach facilitates the study of apoptotic pathways in mammalian systems.
Related Concept Videos
01:15Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
01:24Caspases