Recombinant Human TC-PTP (aa 2-314) Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Assay Buffer: 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mg/mL BSA, 1 mM dithothreitol, pH 7.5
• Recombinant Human TC‑PTP (rhTC-PTP) (Catalog # 1930-PT)
• Substrate: Asp-Ala-Asp-Glu-Tyr(PO₃)-Leu-Ile-Pro-Gln-Gln-Gly (Catalog # ES006)
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Costar, Catalog # 92592) or equivalent
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
• Phosphate Standard: 1 M KH₂PO₄

1. Dilute rhTC-PTP to 0.025 µg/mL in Assay Buffer.
2. Load 40 µL of diluted rhTC-PTP to a plate. Include Substrate Blanks containing 40 µL Assay Buffer in place of rhTC-PTP.
3. Dilute the Substrate in Assay Buffer to 1 mM 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 1M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of assay buffer 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 dilution to use as a standard.) Perform six additional one-half serial dilutions of the 100 µM Phosphate stock. The standard curve has a range of 0.078 to 5 nmol per well.
6. Load 50 µL of the standards and blanks to the plate.
7. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
8. Add 10 µL of the Malachite Green Reagent B to all wells. 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.

• rhTC-PTP: 0.000001 mg (1 ng)
• Substrate: 200 µM