Recombinant Human PTP1B Protein, CF

• Assay Buffer: 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mg/mL BSA, 1 mM dithiothreitol, pH 7.5
• Recombinant Human PTP1B (rhPTP1B) (Catalog # 1366-PT)
• Substrate: Asp-Ala-Asp-Glu-Tyr(PO₃)-Leu-Ile-Pro-Gln-Gln-Gly (Catalog # ES006), 10 mM stock in diH₂O
• 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 rhPTP1B to 0.0625 μg/mL in Assay Buffer.
2. Load 40 µL of diluted rhPTP1B to a 96 well clear plate with 40 µL Assay Buffer as blanks.
3. Dilute the Substrate to 500 µM and add 10 µL to the wells.
4. Cover the plate with parafilm or a plate sealer and incubate at 30 °C for 30 minutes.
5. Prepare a standard curve from the 1 M Phosphate Standard supplied in the Malachite Green Phosphate Detection Kit.
1. Add 10 μL of the 1 M Phosphate Standard to 990 μL of Assay Buffer for a 10 mM stock.
2. Add 10 μL of the 10 mM phosphate stock to 990 μL of Assay Buffer for a 100 μM stock. (This is the first dilution to use as a standard).
3. Perform six additional 1:2 serial dilutions of the 100 μM phosphate stock. The standard curve has a range of 0.078 to

   5.0 nmol per well.

6. After the 30 °C incubation, transfer (in duplicate) 50 μL of the standards and blanks (Assay Buffer) to the plate.
7. Add 10 μL of the Malachite Green Reagent A to each sample, standard, and blank. Mix and incubate for 10 minutes at room temperature.
8. Add 10 μL of the Malachite Green Reagent B to each sample, standard, and blank. Mix and incubate for 20 minutes at room temperature.
9. Read plate at 620 nm (absorbance) in endpoint mode.
10. Calculate specific activity:

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

Per Well:

• Phosphate standard curve: 5.0, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0 nmoles
• rhPTP1B: 0.0000025 mg
• Substrate: 71.4 μM