Cathepsin B Activity Assay Kit (Fluorometric)

Cathepsin B is a cysteine protease, of the C1 family of papain-like peptidases, that primarily functions as an endopeptidase within endo-lysosomal compartments in normal cells. Cathepsin B is closely linked to apoptosis, with activated caspase-8 leading to the release of cathepsin B to the cytosol, which in turn leads to cytochrome c release and apoptosis. Overexpression of Cathepsin B has been shown to correlate with metastatic potential and the enzyme is implicated in the ability of tumor cells to invade the extracellular matrix and to metastasize to secondary sites. Cathepsin B levels are also markedly elevated in a variety of neurological disorders including Alzheimer's disease (AD).

Cathepsin B Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the activity of Cathepsin B in cell lysates, plasma, serum, urine, saliva and other biological liquid samples. In this assay, Cathepsin B cleaves a synthetic specific substrate releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 360/460 nm. The increase in fluorescence intensity is directly proportional to Cathepsin B activity in the sample.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 360/460 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 B 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=360/460 nm continuously at 37°C and record data every 5 mins for 30 mins.