|View Universal Kinase Activity Assay Principle|
The Universal Kinase Activity Kit (Catalog # EA004) provides a simple, non-radioactive, high-throughput compatible format for assaying the enzyme activity of kinases in vitro. The majority of kinases use ATP as the phosphate donor. They transfer the terminal phosphate group of ATP to a substrate, producing ADP as a by-product. This kit is an ADP-based phosphatase-coupled kinase assay that utilizes CD39L2/ENTPD6 as a coupling phosphatase. CD39L2 releases the beta-phosphate from ADP, and the released inorganic phosphate is detected using malachite green reagents. The amount of inorganic phosphate released is proportional to the amount of ADP generated during the kinase reaction and thus, reflects the kinetics of the reaction.
For accurate kinase activity determination, the amount of ADP produced in the kinase reaction needs to be known. It can be determined using the concentration of inorganic phosphate released from ADP and the coupling rate of the kinase reaction. The coupling rate is defined as the percentage of the ADP product that has been converted to the signal of free inorganic phosphate by the coupling reaction. Coupling rates are dependent on the rate constant, reaction volume, and the reaction time. Coupling rates for a 10 minute, 20 minute, and 30 minute kinase reaction performed using varying amounts of ATP and CD39L2 are provided in the tables below.
If conditions of your experiment, such as pH, temperature, NaCl or Ca2+ concentrations, are different from the conditions used for the tables, the coupling rate can be calculated. Please see the protocol in Technical Resources to determine the rate constant and calculate the coupling rate.
For more detailed technical information, please also see: Wu, Z.L. (2011) PLoS ONE 6(8):e23172.
View Larger Image
The activity of Recombinant Human PKA C beta (Catalog # 4596-KS) was assayed using the Universal Kinase Activity Kit (Catalog # EA004) with 0.1 μg of CD39L2 in 50 μL for 10 minutes. The corrected optical density was plotted against kinase concentration. The slope of the line was determined to be 0.908 OD/μg. Using a phosphate conversion factor of 3504 pmol/OD (previously determined), the specific activity was calculated to be 318 pmol/min/μg. After correction with the coupling rate 0.475, the activity is 670 pmol/min/μg.