Recombinant Human Carnosine Dipeptidase 1 Protein, CF

• Assay Buffer: 50 mM Tris, pH 7.5
• Recombinant Human Carnosine Dipeptidase 1/CNDP1 (rhCPGL2) (Catalog # 2489-ZN)
• Substrate: L-Carnosine (Sigma, Catalog # C9625), 100 mM stock in deionized water
• Trichloroacetic acid (TCA) (Sigma, Catalog # T6399), 10% in deionized water
• NaOH, 2 M stock in deionized water
• o-Phthaldialdehyde (OPA) (Sigma, Catalog # P0657), 50 mg/mL in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCDNP1 to 2 ng/µL in Assay Buffer.
2. Dilute Substrate to 2 mM in Assay Buffer.
3. Mix 50 μL of 2 ng/µL rhCDNP1 and 50 μL 2 mM Substrate for a final concentration of 1 ng/µL and 1 mM respectively. Include a Substrate Blank containing 50 μL rhCDNP1, 50 μL 1% TCA added 1 minute prior to substrate addition, and 50 μL Substrate.
4. Incubate for 1 hour at room temperature.
5. Stop reaction by adding 50 μL 1% TCA (except the Substrate Blank). Centrifuge for 10 minutes at 13,000 rpm in a microcentrifuge to spin down any precipitate.
6. Transfer the supernatant to new tubes.
7. Dilute OPA to 5 mg/mL in 2 M NaOH.
8. Add 50 μL of 5 mg/mL OPA to each reaction.
9. Incubate for 30 minutes at room temperature.
10. Load 200 µL (entire volume) of the incubated samples into the plate.
11. Read at excitation and emission wavelengths of 360 nm and 460 nm (top read), respectively, in endpoint mode.
12. Calculate Specific Activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard L-Histidine (Sigma, Catalog # H6034).

• rhCDNP1: 0.100 µg
• Substrate: 0.5 mM