Recombinant S. cerevisiae PPA1 Protein, CF

• Assay Buffer: 25 mM MES, 8 mM MgCl₂, pH 7.0
• Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (ryPPA1) (Catalog # 8088-PP)
• Substrate: Sodium Pyrophosphate (Sigma, Catalog # P8010), 125 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.
2. Prepare the standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer.  The standard curve has a range of 0.039 to 2.5 nmol per well.
3. Dilute Substrate to 1 mM in deionized water.
4. Dilute ryPPA1 to 0.01 µg/mL in Assay Buffer.
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 0.01 µg/mL ryPPA1 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
7. Start the reaction by adding 25 µL of 1 mM Substrate to the wells, excluding the standard curve and curve blank.
8. Incubate sealed plate 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 Substrate Blank.
     ** Note that 2 mol of phosphate are produced for every 1 mol of Pyrophosphate consumed.

• ryPPA1: 0.00025 µg
• Sodium Pyrophosphate: 0.5 mM