Azoreductase/NQO1 Protein, Human (His)

Materials
Assay buffer: 50 mM HEPES, 0.2 M NaCl, 5 μM FAD, 0.05% Tween? 20, pH 7.5
Azoreductase/NQO1 Protein, Human (His) (HY-P74405)
Substrates: Resazurin (HY-111391), NADH
Standard: Resorufin (HY-123533A)

Procedure
1. Dilute Resorufin with assay buffer to the following concentrations: 0, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 μM.
2. Add 100 μL of each concentration of standard into wells.
3. Measure the Excitation/emission values at 540 nm and 585 nm using a microplate reader.
4. Plot the standard curve with the amount of standard on the x-axis and the measured RFU on the y-axis to obtain the standard curve equation.
5. First, reconstitute the Human NQO1 protein with pure water to 100 μg/mL, then dilute it to 0.0075 μg/mL with assay buffer.
6. Dilute NADH to 800 μM in the assay buffer.
7. Dilute Resazurin to 40 μM in assay buffer.
8. Mix equal volumes of diluted NADH and Resazurin to prepare the substrate mixture. Use immediately after mixing.
9. Add 50 μL of 0.0075 μg/mL Human NQO1 into the plate, then add 50 μL of substrate mixture to start the reaction. For the blank group, add 50 μL of assay buffer and 50 μL of substrate mixture.
10. Measure the Excitation/emission values at 540 nm and 585 nm using a microplate reader.
11. Calculate Specific Activity: