Recombinant Human IDO2 Protein, CF

• Assay Buffer: 50 mM Tris, 20% Glycerol, pH 7.5
  
• 0.405 M Tris, pH 8.0
• Recombinant Human IDO2 (rhIDO2) (Catalog # 9967-AO)
• Ascorbic Acid (Sigma, Catalog # 255564), 500 mM stock in deionized water
• L-Tryptophan (Sigma, Catalog # T0254), 40 mM stock in deionized water
• Catalase (Sigma, Catalog # C30), 100,000 units/mL stock diluted in 50 mM MES, pH 6.5
• Methylene Blue (Sigma, Catalog # 28514), 10 mM stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare the Substrate Mixture.
        a.   Dilute Ascorbic Acid to 80 mM in 0.405 M Tris, pH 8.0
        b.   Prepare a mixture of 9000 units/mL Catalase and 40 µM Methylene Blue in Assay Buffer.
        c.   Mix equal volumes of 1a and 1b for final concentrations of 40 mM Ascorbic Acid, 4500 units/mL Catalase and 20 µM Methylene
        Blue.
2. Dilute rhIDO2 to 160 ng/µL in Assay Buffer.
3. Load 25 µL of 160 ng/µL of rhIDO-2 to a clear plate, and start the reaction by adding 25 µL of 40 mM L-Tryptophan, followed by 50 µL of Substrate Mixture.  Include a Substrate Blank containing 25 µL of Assay Buffer, 25 µL of L-Tryptophan and 50 µL of Substrate Mixture.
4. Read in kinetic mode for 5 minutes at an absorbance of 321 nm.
5. Calculate specific activity:

*Adjusted for Substrate Blank
**Using the extinction coefficient 3750 M^-1cm^-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD

Per Well:

• rhIDO2: 4.0 μg
• Ascorbic Acid: 20 mM
• L-Tryptophan: 10 mM
• Catalase: 225 units
• Methylene Blue: 10 µM