Recombinant Human alpha-Aminoadipate Aminotransferase, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 20 mM HEPES, 50 mM NaCl, pH 8.0
• Recombinant Human alpha ‑Aminoadipate Aminotransferase (rhAADAT) (Catalog # 7927-AT)
• L-Kynurenine (Sigma, Catalog # K8625), 40 mM stock in 80 mM HEPES, 8 mM HCl, pH 8.0
• alpha -Ketoglutarate (Sigma, Catalog # K2010), 1 M stock in diH₂O
• Pyridoxal 5’-phosphate (Sigma, Catalog # P9255), 100 mM stock in 1 M HEPES, pH 8.0
• KYNA Development Mixture: 2 M ZnCl₂, 2 M NaOAc in diH₂O
• Standard: Kynurenic acid (Catalog # 0223), 50 mM stock in 100 mM NaOH
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute 50 mM Kynurenic acid standard to 400 µM in Assay Buffer. This is the first point of the standard curve.
2. Perform six additional one-half serial dilutions in Assay Buffer. The standard curve has a range of 156‑10,000 pmol per well.
3. Dilute rhAADAT to 5 µg/mL in Assay Buffer.
4. Prepare Substrate Component Solution containing 50 mM HEPES, 20 mM alpha -ketoglutarate, 80 µM Pyridoxal 5’-phosphate, pH 8.0.
5. Immediately before use, prepare Substrate Mixture by combining 25 µL of Kynurenine with 50 µL of Substrate Component Solution, per well assayed.
6. Load 25 µL of each standard curve dilution into empty wells of a black well plate.
7. Load 25 µL of dilute rhAADAT into empty wells. Include a Blank containing 25 µL of Assay Buffer. This will serve as both enzyme and standard curve blank.
8. Add 75 µL of Substrate Mixture to all wells used.
9. Cover the plate with a plate sealer and incubate at room temperature for 30 minutes.
10. Add 100 µL of KYNA Development Mixture to all wells used.
11. Read plate at excitation and emission wavelengths of 338 and 405 nm, respectively, in endpoint mode.
12. Calculate specific activity:

     *Derived from the Kynurenic acid curve using linear fitting and adjusted for Blank.

• rhAADAT: 0.125 µg
• alpha -ketoglutarate: 10 mM
• Pyridoxal 5’-phosphate: 40 µM
• Kynurenine: 10 mM