Cathepsin B His tag Protein, Human

Cathepsin B belongs to a family of lysosomal cysteine proteases known as the cysteine cathepsins and plays an important role in intracellular proteolysis. In humans, cathepsin B is encoded by the CTSB gene. Cathepsin B is upregulated in certain cancers, in pre-malignant lesions, and in various other pathological conditions. Cathepsin B may enhance the activity of other proteases, including matrix metalloproteinase, urokinase (serine protease urokinase plasminogen activator), and cathepsin D, and thus it has an essential position for the proteolysis of extracellular matrix components, intercellular communication disruption, and reduced protease inhibitor expression. Cells may become carcinogenic when cathepsin B is unregulated. Cathepsin B has been proposed as a potentially effective biomarker for a variety of cancers. Overexpression of cathepsin B is correlated with invasive and metastatic cancers.

Assay protocol
Principle: Measured by its ability to cleave the fluorogenic peptide substrate, Z-LR-AMC.
Materials
1.Protein Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.0
2.Assay Buffer: 25 mM MES,0.02% Brij-35, pH 5.0
3.Human Cathepsin B, His tag)
4.Substrate: Z-LR-AMC (ES008) 
5.96 ELISA Removable Plate, Black, High binding (GENEVER, Catalog # GMO2-96H)
6.Plate Reader (PerkinElmer, excitation 380 nm and emission 460 nm)
Produce
1.Dilute Cathepsin B to 10 μg/mL in Activation Buffer.
2.Incubate at room temperature for 15 minutes.
3.Dilute Cathepsin B to 0.4, 0.2 and 0.1 μg/mL in Assay Buffer.
4.Dilute substrate to 80 μM in Assay Buffer.
5.Load 50 μL of the 0.4, 0.2 and 0.1 μg/mL Cathepsin B in a black well plate, and start the reaction by adding 50 μL of 80 μM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL of 80 μM Substrate.
6.Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes. Calculate specific activity:

*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino-4-Methyl Coumarin