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A sensitive, continuously recording fluorogenic assay for calpain
S K Mallya1, S Meyer, D Bozyczko-Coyne
1Cephalon Inc., 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
Biochemical and Biophysical Research Communications
|July 24, 1998
Summary
We developed a sensitive fluorogenic assay for thiol protease calpain. This method detects 10 picomolar calpain, offering enhanced sensitivity for inhibitor screening and mechanistic studies.
Area of Science:
- Biochemistry
- Enzymology
- Protease Assays
Background:
- Calpains are cysteine proteases involved in various cellular processes.
- Existing assays for calpain activity may lack sensitivity or continuous recording capabilities.
Purpose of the Study:
- To develop a highly sensitive, continuously recording fluorogenic assay for calpain.
- To enable efficient screening of calpain inhibitors and facilitate mechanistic studies.
Main Methods:
- Utilized the dipeptide substrate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA).
- Performed assay in Tris buffer (pH 7.5) with 0.1% CHAPS.
- Adapted the assay for a 96-well plate format.
Main Results:
- The assay demonstrated linearity over a wide enzyme concentration range.
- Achieved detection of 10 picomolar calpain, surpassing previous methods in sensitivity.
- Observed linear reaction rates for over ten minutes, suitable for kinetic analyses.
Conclusions:
- The developed fluorogenic assay provides a sensitive and robust method for calpain detection.
- This assay facilitates high-throughput screening of calpain inhibitors and detailed mechanistic investigations.