Cathepsin D is a lysosomal aspartyl protease, of the peptidase A1 family. Cathepsin D performs a variety of functions in the cell, including recycling protein through autophagy, maturation of proteins by cleaving precursor proteins and apoptosis. Cathepsin D has a dual role with respect to apoptosis. On the one hand, under conditions of cellular stress such as DNA damage or oxidative stress, Cathepsin D is released from the lysosomes and activates apoptotic pathways by cleaving pro-apoptotic proteins. On the other hand, Cathepsin D can prevent apoptosis by regulating the stability and activity of anti-apoptotic proteins, inhibiting apoptotic pathways, enhancing cellular survival and tissue protection.

Cathepsin D Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the activity of Cathepsin D in cell lysates, plasma, serum, urine, saliva and other biological liquid samples. In this assay, Cathepsin D cleaves a synthetic specific substrate releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 320/405 nm. The increase in fluorescence intensity is directly proportional to Cathepsin D activity in the sample.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 320/405 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, positive control, substrate background to the 96-well microtiter plate.
2. Add 10 µL assay buffer to each well.
3. Add 100 µL of standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 50 µL diluted Cathepsin D substrate solution to each well. Do NOT add to standard wells. 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=320/405 nm continuously at 37°C and record data every 5 min for 30 min.