Recombinant Human DEP-1/CD148 (aa 997-1337) Protein, CF

• Assay Buffer: 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mg/mL BSA, 1 mM dithiothreitol (DTT), pH 7.5
• Recombinant Human DEP‑1/CD148 aa 997‑1337  (rhDEP-1) (Catalog # 1934-DP)
• 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)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhDEP-1 to 0.00625 µg/mL in Assay Buffer.
2. Load 40 µL of diluted rhDEP-1 to a plate. Include 40 µL of Assay Buffer as a Substrate Blank.
3. Dilute Substrate to 1 mM and add 10 µL to all wells. The Substrate concentration is now 200 µM per well.
   Cover the plate with parafilm or a plate sealer and incubate at 30 °C for 30 minutes.
4. Prepare a standard curve from the 1 M 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 standard stock. The standard curve has a range of 0.078 to 5 nmol per well.
5. After the 30 °C incubation, transfer 50 µL of the standards to the plate. Include a blank for the standard curve (50 µL Assay Buffer).
6. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
7. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
8. Read plate at 620 nm (absorbance) in endpoint mode.
9. Calculate specific activity:

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

Per Well:

• rhDEP-1: 0.00000025 mg
• Substrate: 143 µM