Recombinant B. pertussis Adenylate Cyclase 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.

• Assay Buffer: 25 mM Tris, 10 mM MgCl₂, 50 µM CaCl₂, pH 7.0
• Recombinant Bacterial Adenylate Cyclase (rB.Adenylate Cyclase) (Catalog # 8270-AC)
• Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (ryPPA1) (Catalog # 8088-PP)
• Calmodulin (Sigma, Catalog # P1431), 0.168 mg/mL stock in 20 mM Tris, pH 7.5, 10 mM MgCl₂, 1 mg/mL BSA, and 0.1 mM CaCl₂
• ATP (Sigma, Catalog # A7699), 10 mM stock in deionized water
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
2. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
3. Prepare Reaction Mixture containing 1 mM ATP and 10 µg/mL ryPPA1 in Assay Buffer.
4. Dilute Calmodulin to 0.0667 µg/mL in Assay Buffer.
5. Dilute rB.Adenylate Cyclase to 0.02 µg/mL in Assay Buffer.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
7. Load 15 µL of the 0.02 µg/mL rB.Adenylate Cyclase into empty wells of the plate.
8. Add 15 µL of the 0.0667 µg/mL Calmodulin to wells containing enzyme, excluding the standard curve and curve blank. Create a Control by replacing Calmodulin with Assay Buffer.
9. Start the reactions by adding 20 µL of Reaction Mixture all the wells, excluding the standard curve and curve blank.
10. Seal the plate and incubate at room temperature for 10 minutes.
11. Add 30 µL of the Malachite Green Reagent A to all wells.
12. Add 100 µL of deionized water to all wells. Mix briefly.
13. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
14. Read plate at 620 nm (absorbance) in endpoint mode.
15. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

• rB. Adenylate Cyclase: 0.0003 µg
• ryPPA1: 0.2 µg
• Calmodulin: 0.001 µg
• ATP: 0.4 mM