Recombinant Human PADI4 Protein, CF

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

1. Prepare Color Developing Reagent A (80 mM DAMO, 2 mM Thiosemicarbazide) in deionized water.
2. Prepare Color Developing Reagent B (17.35% (v/v) Phosphoric Acid, 33.7% (v/v) Sulfuric Acid, 0.765 mg/mL Ammonium Iron Sulfate) in deionized water.
3. Dilute rhPADI4 to 4 ng/µL in Assay Buffer.
4. Dilute BAEE to 10 mM in Assay Buffer.
5. Dilute 20 mM L-Citrulline stock to 1000 µM in Assay Buffer. This is the first point of the standard curve.
6. Continue standard curve by performing six one-half serial dilutions of the 1000 µM L-Citrulline in Assay Buffer. The standard curve has a range of 0.625 to 40 nmol per well.
7. Pipet 150 µL of each point of the standard curve into microtubes.
8. Combine 75 µL of 4 ng/µL rhPADI4 with 75 µL of 10 mM BAEE in microtubes. Include a Substrate Blank containing Assay Buffer in place of rhPADI4.
9. Incubate reaction, blank, and standard curve for 30 minutes at 37 °C.
10. Prepare Color Developing Reagent C (1 part Reagent A : 3 parts Reagent B).
11. Add 600 μL of Color Developing Reagent C to reaction, blank, and standard curve tubes.
12. Heat the tubes at 95‑100 °C in a heating block for 15 minutes.
13. Cool tubes at room temperature for 5 minutes.
14. Load 200 μL into wells.
15. Read plate at 540 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

     *Derived from the L-Citrulline standard curve using linear fitting and adjusted for Substrate Blank.

• rhPADI4: 0.08 μg
• BAEE: 1 mM