| Assay Method Information | |
| | Inhibitory Effect of Compounds on Cat K |
| Description: | Cathepsin K (CTSK, EC 3.4.22.38) is a lysosomal cysteine protease, participates in osteoclastic bone remodeling and resorption, and can further degrade collagen, gelatin and elastin. The cathepsin K inhibitor screening kit from Biovision uses the ability of the active cathepsin K to cleave a synthetic AFC-based peptide substrate to release AFC and the AFC can be easily quantified by using a fluorometer or fluorescent microplate reader. In the presence of a specific cathepsin K specific inhibitor, the cleavage of the substrate is reduced/eliminated, resulting in a reduction or complete loss of AFC fluorescence. The simple and high-throughput adaptive assay kit can be used to screen/study/characterize a potential inhibitor of the cathepsin K.Figure US20250145576A1-20250508-C00016[0223]20 μL of a buffer, a Cat K inhibitor and test compounds (1 μM and 10 μM) were added to 96-well plates to serve as an EC well, an IC well and an S well, respectively; 50 μL of a Cat K enzyme solution was added into all the wells respectively and the mixture was incubated for 10-15 min at room temperature to establish an enzyme inhibitor complex; 30 μL of a Cat K substrate solution was added into all the wells and the mixture was incubated for 30-60 min at room temperature; and the final volume of the reaction was 100 μL. At 30-60 min, two time points T1 and T2 were selected to detect fluorescence absorption values (Ex/Em=400/505 nm), the fluorescence absorption values were recorded as RFU1 and RFU2, and the inhibition rates (%) of the test compounds on the Cat K were calculated.Slope=(RFU2-RFU1)/(T2-T1)Inhibitionrate(%)=(ECslope-Sslope)/ECslope×100 |
| Affinity data for this assay | |
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