Recently I bought some Cathepsin B protein (HY-P70830), I am looking for reliable methods to activate and measure the activity of Cathepsin B. If anyone has relevant experience or can point me to pertinent literature on this topic, I would greatly appreciate your insights and recommendations!
Lucian Miles
Dilute Cathepsin B to 10 µg/mL in Activation Buffer. Incubate at room temperature for 15 minutes. Dilute Cathepsin B further to 0.2 ng/µL in Assay Buffer. Dilute the substrate to 20 µM in Assay Buffer. In a black 96-well plate, load 50 µL of 0.2 ng/µL Cathepsin B per well. Start the reaction by adding 50 µL of 20 µM substrate. Include a substrate blank: 50 µL Assay Buffer + 50 µL of 20 µM substrate (no enzyme). Measure fluorescence in kinetic mode for 5 minutes at Ex: 380 nm, Em: 460 nm (top read).
Hi Marine Ma
You may refer to the following protocol for Cathepsin B activation and activity assay (also supported by a recent study, PMID: 40175391):
- Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.0
- Assay Buffer: 25 mM MES, pH 5.0
- Fluorogenic Peptide Substrate VII: Z-Leu-Arg-AMC (HY-142021)
Procedure:
The answer to this question is provided by MedChemExpress Technical Support.
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