Recombinant Mouse Cystatin B Protein, CF

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

• Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
• Assay Buffer: 50 mM Tris, pH 7.0
• Recombinant Mouse Cystatin B (rmCystatin B) (Catalog # 1409-PI)
• Papain (Sigma, Catalog # P4762)
• Substrate: Z-Phe-Arg-AMC (Catalog # ES009), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute Papain to 100 µg/mL in Activation Buffer.
2. Incubate at room temperature for 15 minutes.
3. Prepare a curve of rmCystatin B (MW: 11,991 Da) in Assay Buffer. Make the following serial dilutions: 800, 400, 200, 150, 100, 50, 25 and 12.5 nM.
4. Dilute activated Papain to 2.4 µg/mL in Assay Buffer.
5. Combine 20 µL of 2.4 µg/mL Papain with 20 µL of rmCystatin B curve dilutions. Include two controls containing 20 µL Assay Buffer with 20 µL of 2.4 µg/mL Papain.
6. Incubate at room temperature for 10 minutes.
7. After incubation, dilute the rmCystatin B curve 5-fold by adding 160 µL of Assay Buffer.
8. Dilute Substrate to 200 µM in Assay Buffer.
9. In a plate, load 50 µL of the diluted rmCystatin B curve, and the start the reaction by adding 50 µL of 200 µM Substrate to wells.
10. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
11. Derive the 50% inhibition concentration (IC₅₀) value for rmCystatin B by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
12. The specific activity for Papain at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

• Papain: 0.012 µg
• Substrate: 100 µM
• rmCystatin B curve: 40, 20, 10, 7.5, 5, 2.5, 1.25 and 0.625 nM