Recombinant Human Cathepsin A/Lysosom Carboxypeptidase A, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Activation Buffer: 25 mM MES, 5 mM DTT, pH 6.0
• Assay Buffer: 25 mM MES, 5 mM DTT, pH 5.5
• Recombinant Human Cathepsin A/Lysosomal Carboxypeptidase A (rhCathepsin A) (Catalog # 1049-SE)
• Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
•  E 64 (Tocris Catalog # 5208) , 50 mM stock in DMSO
• Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin A to 100 µg/mL in Activation Buffer.
2. Dilute rhCathepsin L to 10 µg/mL in Activation Buffer.
3. Combine equal volumes of rhCathepsin A and rhCathepsin L for final concentrations of 50 µg/mL and 5 µg/mL, respectively.
4. Incubate reaction at 37 °C for 30 minutes.
5. Stop reaction by adding E-64 to a final concentration of 10 µM.
6. Dilute activated rhCathepsin A to 2.0 ng/µL in Assay Buffer.
7. Dilute Substrate to 20 µM in Assay Buffer.
8. Load 50 µL of 2.0 ng/µL rhCathepsin A into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
9. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
10. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)

• rhCathepsin A: 0.1 µg
• Substrate: 10 µM