Cathepsin G is a neutrophil serine protease, stored in cytoplasmic granules and secreted into the extra- and pericellular space upon neutrophil activation. Cathepsin G plays important role in bacterial defense. It acts in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. Besides bactericidal effect, Cathepsin G regulates the inflammatory response by specifically altering the function of cytokines and chemokines. Because of its role in host defense, Cathepsin G is of interest as a potential therapeutic target.

Cathepsin G Activity Assay Kit provides a simple, reproducible, and sensitive tool for measuring Cathepsin G activity in serum, plasma, cell lysates, urine, saliva and other biological fluid samples. This assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate. The light emitted from pNA can be quantified using a spectrophotometer or microplate reader at 405 nm. Then, Cathepsin G activity can be calculated as nmol of pNA released per minute per milliliter of the sample. Because the substrate is also cleaved by chymotrypsin, mast-cell chymase, and some other chymotrypsin-like proteases. A Cathepsin G Inhibitor is included in this assay, the inhibitor-sensitive fraction gives you Cathepsin G-specific activity in 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
• 5 mL conical tubes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 40 µL of samples and positive control to the 96-well microtiter plate.
2. Each sample replicate requires two paired wells, in one sample well, add 10 µL of the Cathepsin G Inhibitor. In the other sample well, add 10 µL of assay buffer so the final volume in each well is 50 µL. Incubate the samples for 10 minutes at 37°C in the dark with mixing.
3. Add 100 µL of each standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 50 µL diluted Cathepsin G substrate solution to each well. Do NOT add to standard wells. Carefully shake the plate for a few seconds to mix well.
5. For end-point reading: Immediately read and record absorbance at 405 nm, incubate the reaction for 30 to 60 min at 37°C protected from light, read and record absorbance again at 405 nm.
   For kinetic reading: Immediately start measuring absorbance at 405 nm continuously at 37°C and record data every 5 minutes for 30 to 60 min, protect from light.