Recombinant Mouse BACE-1 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.

• Assay Buffer: 50 mM Sodium Acetate, 100 mM NaCl, pH 4.0
• Recombinant Mouse BACE-1 (rmBACE-1) (Catalog # 2976-AS)
• Substrate: MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(DNP)-Arg-Arg-NH₂ (Catalog # ES004)
• Heparin (Sigma, Catalog # H3393), 20 mg/mL in deionized water
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmBACE-1 to 20 ng/µL in Assay Buffer.
2. Dilute Heparin to 4 ng/μL in Assay Buffer.
3. Combine equal volumes diluted Heparin and rmBACE-1.
4. Incubate at 37 °C for 30 minutes.
5. Dilute Substrate to 20 μM in Assay Buffer.
6. Load 50 µL of the rmBACE-1/heparin mixture in 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.
7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Calculate specific activity:

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

• rmBACE-1: 0.500 µg
• Substrate: 10 µM
• Heparin: 1 ng/μL