Recombinant Human PRL-2/PTP4A2 Protein, CF

• Assay Buffer: 50 mM HEPES, 10 mM DTT, pH 7.5
• Recombinant Human PRL-2/PTP4A2 (rhPRL-2) (Catalog # 6694-PT)
• Substrate: p-Nitrophenyl phosphate (Sigma, Catalog # N2765), 10 mM stock in deionized water
• NaOH, 0.2 M in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhPRL-2 to 40 µg/mL in Assay buffer.
2. Dilute Substrate to 5 mM in Assay buffer.
3. Prepare reaction mixtures by combining equivalent volumes of dilute rhPRL-2 and dilute Substrate in microtubes. Prepare an Enzyme Control by combining diluted rhPRL-2 with twice the volume of 0.2 M NaOH, mix briefly, then add a volume of Substrate equivalent to volume of rhPRL-2. The Enzyme Control will have 2x the volume of the reaction mixture.
4. Incubate reactions and controls at 37 °C for 24 hours.
5. Load 100 µL of Reactions into a plate in triplicate and stop the reactions by adding 100 µL 0.2 M NaOH.
6. Load 200 µL of Enzyme Controls into a plate in triplicate.
7. Read plate at 410 nm (absorbance) in endpoint mode.
8. Calculate specific activity:

     *Adjusted for Enzyme Controls
     **Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326)

Per Well:

• rhPRL-2: 2 µg
• pNPP: 1.25 mM