Caspase-1 (ICE, IL-1β Converting Enzyme) is the prototypical member of the ICE family of cysteine proteases, also known as caspases (cysteinyl aspartate-specific proteases). Caspase 1 was first identified as a novel protease that generates the proinflammatory cytokine, interleukin 1 beta (IL-1 Beta), by cleaving the pro-interleukin after the Asp116 residue. Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. Inhibition of caspases can delay apoptosis, implicating a potential role in drug screening efforts.

Caspase 1 Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the activity of caspase 1 in cell lysates samples. In this assay, caspase 1 cleaves a synthetic specific substrate YVAD releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 400/505 nm. The increase in fluorescence intensity is directly proportional to caspase 1 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 Caspase 1 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 mins for 30 mins.