Recombinant Human PRL-1/PTP4A1 Protein, CF

• Assay Buffer: 50 mM HEPES, 10 mM DTT, pH 7.5
• Recombinant Human PRL-1/PTP4A1 (rhPTP4A1) (Catalog # 8490-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 rhPTP4A1 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 rhPTP4A1 and dilute Substrate in microtubes. Include an Enzyme Control by combining dilute rhPTP4A1 with twice the volume of 0.2 M NaOH, mix briefly, then add a volume of dilute Substrate equivalent to the volume of rhPTP4A1.  The Enzyme Control will have 2x the volume of the reaction mixture.
4. Incubate Reactions and Enzyme 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 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:

• rhPTP4A1: 2 µg
• pNPP: 1.25 mM