Recombinant Mouse IMPAD1 Protein, CF

• Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5
• Recombinant Mouse Inositol Monophosphatase 3/IMPAD1 (rmIMPAD1) (Catalog # 7028-PD)
• Adenosine 3′,5′-diphosphate (PAP) (Sigma, Catalog # A5763), 20 mM stock in deionized water
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute PAP to 1 mM in Assay Buffer.
2. Dilute rmIMPAD1 to 1 µg/mL in Assay Buffer.
3. 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.
4. 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.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 25 µL of the 1 µg/mL rmIMPAD1 into the plate. Include a Control containing 25 µL of Assay Buffer.
7. Start the reaction by adding 25 µL of 1 mM PAP to the wells, excluding the standard curve and curve blank.
8. Cover the plate with parafilm or a plate sealer and incubate at room temperature for 10 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 µL of deionized water to all wells. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
13. Calculate specific activity:

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

Per Reaction:

• rmIMPAD1: 0.025 μg
• PAP: 0.5 mM