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Updated: Jan 31, 2026
11:37
Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
Published on: July 8, 2010
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FRET-Based Assays to Determine Calpain Activity
Christian-Scott E McCartney1, Peter L Davies2
1Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada.
Methods in Molecular Biology (Clifton, N.J.)
|January 9, 2019
Summary
Calpain activity measurement is challenging due to autoproteolysis. A new FRET-based assay accurately quantifies calpain kinetics by monitoring initial substrate cleavage, aiding in inhibitor characterization.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Calpains are calcium-dependent proteases involved in cellular signaling.
- Calpain autoproteolysis complicates accurate kinetic measurements.
- Existing assays often lack the sensitivity for rapid kinetic analysis.
Purpose of the Study:
- To develop a facile and reliable assay for quantifying calpain activity.
- To address the challenges posed by calpain autoproteolysis in kinetic studies.
- To enable the characterization of calpain-specific inhibitors.
Main Methods:
- Utilized FRET peptide substrates with high enzyme-substrate affinity.
- Employed continuous monitoring of FRET fluorescence.
- Developed a variation for high-throughput screening using the calpain protease core.
Main Results:
- The assay accurately measures initial enzyme reaction velocity.
- Demonstrated the ability to overcome autoproteolysis limitations.
- Facilitated the characterization of calpain inhibitors.
Conclusions:
- A robust FRET-based assay provides accurate calpain kinetic data.
- This method is suitable for both detailed kinetic analysis and high-throughput screening.
- The assay aids in the development of calpain-specific therapeutics.
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