Cathepsin B Protein, Rat (HEK293, His)

Cathepsin B is a lysosomal cysteine protease that plays a role in intracellular protein catabolism. Cathepsin B mediates JNK signaling pathway to regulate the migration of glioma initiation cells. Cathepsin B is involved in the pathology of chronic inflammatory diseases of the airway and joints, as well as cancer and pancreatitis. Cathepsin B Protein, Rat (HEK293, His) is the recombinant rat-derived Cathepsin B protein, expressed by HEK293 , with C-His labeled tag.

Cathepsin B is a lysosomal cysteine protease that plays a role in intracellular protein catabolism and is involved in other physiological processes such as antigen processing in immune response, hormone activation, and bone turnover. Cathepsin B mediates JNK signaling pathway to regulate the migration of glioma initiation cells. Cathepsin B can enhance the activity of other proteases, including matrix metalloproteinases, urokinase (serine protease urokinase plasminogen activator), and Cathepsin D. Cathepsin B is involved in the pathology of chronic inflammatory diseases of the airway and joints, as well as cancer and pancreatitis^[1][2][3].

Measured by its ability to cleave the fluorogenic peptide substrate Z-LR-AMC . The specific activity is >2,500 pmol/min/μg. (Activation description: The proenzyme needs to be activated in acid reducing buffer for an activated form.)

Materials
Activation buffer: 25 mM MES, 5 mM DTT, pH 5.0
Assay buffer: 25 mM MES, pH 5.0
Cathepsin B Protein, Rat (HEK293, His) (HY-P74345)
Substrate: Z-Leu-Arg-AMC (HY-142021)
Standard: 7-Amino-4-methylcoumarin (AMC, HY-D0027)

Procedure
1. Dilute AMC to 0, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μmol/L respectively with assay buffer.
2. Add 100 μL of each dilution to black microplate wells.
3. Set excitation and emission wavelengths to 380 nm and 460 nm (top read), and read in kinetic mode for 5 minutes.
4. Plot a standard curve with the measured RFU as the abscissa and the amount of standard substance as the ordinate, and obtain the standard curve equation.
5. Dilute the test protein to 10 μg/mL with activation buffer; dilute Rat Cathepsin B to 100 μg/mL with assay buffer.
6. Incubate at room temperature for 15 minutes.
7. Dilute Rat Cathepsin B to 0.2 μg/mL with assay buffer.
8. Dilute the substrate to 20 μM with assay buffer.
9. Add 50 μL of the test protein at each concentration to the assay wells, and initiate the reaction by adding 50 μL of 20 μM substrate. For blank wells, add 50 μL of assay buffer and 50 μL of 20 μM substrate without protein.
10. Set excitation and emission wavelengths to 380 nm and 460 nm (top read), and read in kinetic mode for 5 minutes.
11. Calculate the specific activity:

Rat

HEK293

C-6*His

Q6IN22 (H18-F339)

64529  [NCBI]

Full Length of Mature Protein

HDKPSFHPLSDDMINYINKQNTTWQAGRNFYNVDISYLKKLCGTVLGGPKLPERVGFSEDINLPESFDAREQWSNCPTIAQIRDQGSCGSCWAFGAVEAMSDRICIHTNGRVNVEVSAEDLLTCCGIQCGDGCNGGYPSGAWNFWTRKGLVSGGVYNSHIGCLPYTIPPCEHHVNGSRPPCTGEGDTPKCNKMCEAGYSTSYKEDKHYGYTSYSVSDSEKEIMAEIYKNGPVEGAFTVFSDFLTYKSGVYKHEAGDVMGGHAIRILGWGIENGVPYWLVANSWNVDWGDNGFFKILRGENHCGIESEIVAGIPRTQQYWGRF

Approximately 35-43 kDa due to the glycosylation.

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

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of 25 mM Tris, 150 mM NaCl, pH 7.5. or PBS, pH 7.4.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

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.