The calpains are a family of intracellular cysteine proteases, which consists of at least 15 ubiquitous and tissue-specific isoforms. The best-characterized calpains are the isoforms µ- and m-calpain, which are also known as calpain 1 and calpain 2, respectively. Calpains are calcium-dependent and function by modulating the biological activities of many proteins through selective cleavage. Studies have demonstrated that calpains are implicated in a variety of calcium-regulated cellular processes such as signal transduction, cell proliferation, differentiation, cell cycle progression, apoptosis, and platelet activation. Deregulation of calpains activities has been implicated in various pathological phenomena such as atherosclerosis, Alzheimer’s disease, diabetes, and cancer. Calpains represent potential therapeutic targets for drug development.

Calpain Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the activity of calpain in cell lysates samples. In this assay, calpain cleaves a synthetic specific substrate LLY releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 400/505 nm. The increase in fluorescence intensity is directly proportional to calpain activity in the sample.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 400/505 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Incubator at 37°C
• Tubes to prepare sample dilutions
• 15 mL and 50 mL conical tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 40 µL of samples to the 96-well microtiter plate. Include substrate background wells by adding Assay Buffer only.
2. Add 10 µL Assay Buffer to each well. If adding an inhibitor, additional Assay Buffer is not needed.
3. Add 100 µL of each standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 50 µL diluted Calpain Substrate solution to each well. Carefully shake the plate for a few seconds to mix well.
5. Measure fluorescence signal. For kinetic reading, immediately start measuring fluorescence intensity at Ex/Em=400/505 nm continuously at 37°C and record data every 5 minutes for 30 minutes.