Cathepsin B Protein, Human (HEK293, C-His)

Cathepsin B Protein, Human (HEK293, His) functions in intracellular protein catabolism and in certain situations may also be involved in other physiological processes, such as processing of antigens in the immune response, hormone activation and bone turnover^[1].

Cathepsin B is a lysosomal cysteine protease of the papain family. It functions in intracellular protein catabolism and in certain situations may also be involved in other physiological processes, such as processing of antigens in the immune response, hormone activation and bone turnover. There is also evidence that cathepsin B is implicated in the pathology of chronic inflammatory diseases of airways and joints, and in cancer and pancreatitis^[1].

Measured by its ability to cleave the fluorogenic peptide substrate Z-LR-AMC. Read at excitation and emission wavelengths of 380 nm and 460 nm. The specific activity is >6500 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated in acid reducing buffer for an activated form.)

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  Measured by its ability to cleave the fluorogenic peptide substrate Z-LR-AMC. Read at excitation and emission wavelengths of 380 nm and 460 nm . The specific activity is 8552.8807 pmol/min/µg, as measured under the described conditions.

Materials
Activation buffer: 25 mM MES, 5 mM DTT, pH 5.0
Detection buffer: 25 mM MES, 0.02% Brij-35, pH 5.0
Cathepsin B Protein, Human (HEK293, C-His) (HY-P7993A)
Substrate: Z-Leu-Arg-AMC (HY-142021)
Standard: 7-amino, 4-Methyl Coumarin (AMC, HY-D0027)

Procedure
1. Standard Curve: Dilute AMC in detection buffer to concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2, 6.25, 12.5, 25, 50, and 100 μmol/L, respectively. Add 100 μL of each dilution into black microplate wells. Set the excitation and emission wavelengths to 380 nm and 460 nm, respectively. Plot the standard curve with the measured RFU (Relative Fluorescence Units) on the x-axis and the amount of standard substance on the y-axis to obtain the standard curve equation.
2. Dilute the protein sample to 10 μg/mL in activation buffer.
3. Incubate at room temperature for 15 minutes.
4. Dilute Human Cathepsin B to 0.2 μg/mL in detection buffer.
5. Dilute the substrate to 20 μM in detection buffer.
6. Add 50 μL of each protein concentration into the assay wells and add 50 μL of 20 μM substrate to initiate the reaction. For the blank wells, add 50 μL of detection buffer and 50 μL of 20 μM substrate without protein.
7. Set the excitation and emission wavelengths to 380 nm and 460 nm, respectively. Read the fluorescence in kinetic mode for 5 minutes.
8. Calculate Specific Activity:

Human

HEK293

C-6*His

P07858 (R18-I339)

1508

Full Length of Mature Protein

RSRPSFHPLSDELVNYVNKRNTTWQAGHNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQGSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLTCCGSMCGDGCNGGYPAEAWNFWTRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPTYKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMGGHAIRILGWGVENGTPYWLVANSWNTDWGDNGFFKILRGQDHCGIESEVVAGIPRTDQYWEKI

Approximately 35-45 kDa due to the glycosylation.

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  Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4 or PBS, pH 7.4, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in PBS, pH 7.4. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.