Recombinant Human Renin Protein, CF

• Activation Buffer: 5 mM Tris, 15 mM NaCl, 1 mM CaCl₂, 0.005% Brij-35, pH 7.5
• Assay Buffer:  50 mM Sodium Acetate, 150 mM NaCl, 2 mM EDTA, pH 5.0
• Recombinant Human Renin (rhRenin) (Catalog # 4090-AS)
• Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
• Substrate: Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(dabcyl)-Arg, 1 mM stock in DMSO
• 4-(2-Aminoethyl-benzensulfonyl fluoride hydrochloride) (AEBSF) (Catalog # EI001), 100 mM stock in deionized water.
• Black 96 well Plate
• Plate Reader with Fluorescence Read Capability

1. Dilute rhRenin to 0.2 mg/mL in Activation Buffer.
2. Dilute rhTrypsin 3 to 4 µg/mL in Activation Buffer.
3. Mix equal volumes of the diluted rhRenin and Trypsin.
4. Incubate at 37 °C for 1 hour to activate rhRenin.
5. Stop activation with AEBSF at 1 mM and incubate at room temperature for 30 min.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Dilute rhRenin to 20 µg/mL in Assay Buffer.
8. Load into a black well plate 50 µL of 20 µg/mL of rhRenin and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank by combining 50 µL of 20 µM Substrate with 50 µL of Assay Buffer.
9. Read at excitation and emission wavelengths of 350 nm and 490 nm (top read), respectively, in kinetic mode for 5 minutes.
10. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard Fmoc-Glu(EDANS)-OH.

Per Well:

• rhRenin: 1.0 µg
• Substrate: 10 µM