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Published on: January 12, 2016
Quantification of active caspase 3 in apoptotic cells
P A Saunders1, J A Cooper, M M Roodell
1R & D Systems, Inc., Minneapolis, Minnesota 55413, USA.
Analytical Biochemistry
|August 10, 2000
Summary
We developed a new assay to measure active caspase 3 in apoptotic cells. This enzyme-linked immunosorbent assay (ELISA) uses a biotinylated inhibitor to specifically detect active caspase 3, aiding apoptosis research.
Area of Science:
- Biochemistry
- Cell Biology
- Assay Development
Background:
- Caspase 3 is a key executioner caspase in apoptosis.
- Quantifying active caspase 3 is crucial for understanding apoptotic pathways.
- Existing methods may lack specificity or fail to distinguish active from latent forms.
Purpose of the Study:
- To develop and validate a novel enzyme-linked immunosorbent assay (ELISA) for quantifying active caspase 3.
- To differentiate active caspase 3 from latent forms and other caspases.
- To establish a sensitive and specific method for apoptosis research.
Main Methods:
- Utilized a biotinylated inhibitor for covalent modification of active caspase 3 sites.
- Employed an antibody specific for caspase 3 for capture on ELISA plates.
- Detected bound caspase 3 using horseradish peroxidase (HRP)-conjugated streptavidin.
Main Results:
- Quantified 6.6 ng of active caspase 3 per 10^6 apoptotic Jurkat cells.
- Demonstrated assay specificity for caspase 3 against other caspases (2, 7, 8, 10) and in caspase 3-negative cells (MCF-7).
- Confirmed discrimination between active and latent caspase 3 via Western blotting.
Conclusions:
- The developed ELISA provides a specific and sensitive method for quantifying active caspase 3.
- The assay successfully differentiates active from latent caspase 3.
- The underlying principle holds potential for developing similar assays for other enzyme classes.
