Caspases (Cysteine-requiring aspartate protease) are a family of proteases that mediate cell death and are important to the process of apoptosis. Caspase 3 is a member of the CED-3 subfamily of Caspases and is one of the critical enzymes of apoptosis. Caspase 3, which is an effector Caspase, is the most studied of mammalian Caspases. Caspase 3 can process pro-Caspase 2, 6, 7, and 9 and specifically cleave most of the Caspase-related substrates known to date. This cleavage is part of the mechanism leading to cell death. In addition, Caspase 3 plays a central role in mediating nuclear apoptosis, including chromatin condensation and DNA fragmentation, as well as cell blebbing. Caspase 3 activity is tissue, cell type, or death stimulus-specific.

Caspase-3 Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring Caspase-3 Activity in cell lysates. This assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 405 nm. The caspase-3 activity can be calculated as nmol of pNA released per minute per milliliter of the sample.

• Microplate reader capable of measuring absorbance at 405 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C
• 15 mL conical tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of samples, controls, and Background to the 96-well microtiter plate.
2. Add 50 µL of Reaction buffer with DTT to each well, mix well.
3. Add 5 µL of Caspase-3 Substrate solution to each well, mix well.
4. Incubate the plate for 1 hour at 37°C with mixing in the dark.
5. Measure the absorbance at 405 nm using a plate reader