Recombinant Human NQO-1 Protein, CF

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

• Assay Buffer: 50 mM HEPES, 0.2 M NaCl, 5 µM FAD, 0.05% Tween® 20, pH 7.5
• Recombinant Human NQO-1 (rhNQO-1) (Catalog # 7567-DH)
• beta -Nicotinamide Adenine Dinucleotide, reduced (NADH) (Sigma, Catalog # N8129), 20 mM stock in  0.1 M Sodium Borate, pH 9.0
• Resazurin (Catalog # AR002) (MW = 251.17 Da)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhNQO-1 to 0.0075 µg/mL in Assay Buffer.
2. Dilute NADH to 800 µM in Assay Buffer.
3. Dilute Resazurin to 40 µM in Assay Buffer.
4. Combine equivalent volumes of diluted NADH and Resazurin to make the Substrate Mixture. Mix well and use immediately.
5. Load 50 µL of 0.0075 µg/mL rhNQO-1 into a plate, and start the reaction by adding 50 µL of the Substrate Mixture. For a Substrate Blank, load 50 µL of Assay Buffer and 50 µL of Substrate Mixture.
6. Read at excitation and emission wavelengths of 540 nm and 585 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard Resorufin (Sigma, Catalog # R3257).

• rhNQO-1: 0.375 ng
• NADH: 200 µM
• Resazurin: 10 µM