Recombinant Human Thymidine Kinase 1 Protein, CF

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

• Universal Kinase Activity Kit (Catalog # EA004)
• 10X Assay Buffer  (supplied in kit): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl₂, 100 mM CaCl₂ pH 7.0
• Recombinant Human Thymidine Kinase 1 (rhTK-1) (Catalog # 8180-TK)
• Acceptor substrate: Thymidine (Sigma, Catalog # T9250), 100 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 containing 0.5 mM DTT.
2. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
3. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
4. Prepare a substrate mixture containing 0.5 mM ATP and 12.5 mM Thymidine in Assay Buffer.
5. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.
6. Dilute rhTK-1 to 25 µg/mL in Assay Buffer.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
8. Load 20 µL of the 25 µg/mL rhTK-1 into the plate. Include a Control containing 20 µL of Assay Buffer.
9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to the wells, excluding the standard curve.
10. Add 20 µL of substrate mixture to the wells, excluding the standard curve.
11. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.
12. Add 30 µL of 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 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:

     *Derived from the phosphate standard curve using linear fitting and adjusted for Control.

      ** The coupling rate is 0.475 under these conditions.

• rhTK-1: 0.5 µg
• Coupling Phosphatase 4: 0.1 μg
• ATP: 10 nmol (0.2 mM)
• Thymidine: 250 nmol (5 mM)