Recombinant Human PANK1 His-tag Protein, CF

• Universal Kinase Activity Kit (Catalog # EA004)
• 1X Assay Buffer:  25 mM HEPES, 150 mM NaCl, 10 mM MgCl₂, 10 mM CaCl₂, pH 7.0 (10X Assay Buffer in kit EA004)
• Recombinant Human PANK1 (rhPANK1) (Catalog # 11161-PK)
• Pantothenic Acid (Sigma, Catalog # P5155), 20 mM stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare 1X Assay Buffer by diluting 10X stock (Phosphatase Buffer 4 that is provided in kit) 10 fold with deionized water.
2. Prepare a standard curve from the 1 mM Phosphate Standard (provided in kit) by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
4. Prepare a Reaction Mixture containing 0.4 mM ATP and 2 mM Pantothenic Acid in 1X Assay Buffer.
5. Dilute Coupling Phosphatase 4 to 10 µg/mL in 1X Assay Buffer.
6. Dilute rhPANK1 to 33.34 µg/mL in 1X Assay Buffer.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
8. Load 15 µL of the 33.34 µg/mL rhPANK1 into empty wells of the same plate as the curve. Include a Control containing 15 µL of 1X Assay Buffer.
9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
10. Start the reaction by adding 25 µL of Reaction Mixture to all wells, excluding standard curve.
11. Seal and incubate plate at room temperature for 10 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
13. Add 100 µL of deionized water to all wells. Mix briefly.
14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
15. Read plate at 620 nm (absorbance) in endpoint mode.
16. Calculate specific activity:
    
    

• rhPANK1: 0.5 µg
• Coupling Phosphatase 4: 0.1 µg
• ATP: 0.2 mM
• Pantothenic Acid: 1 mM

Scientific Data

Enzyme Activity View Larger

SDS-PAGE View Larger

1 μg/lane of rhPANK1 (Catalog # 11161-PK) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing a band at 40 kDa.