Universal Kinase Activity Kit, 2 Plate

View Universal Kinase Activity Assay Principle

Features of the Universal Kinase Activity Kit

Versatile

• Applicable for all kinase reactions that produce ADP

Convenient

• Non-radioactive
• No time-consuming separation steps (e.g. column chromatography)

Quantitative

• The phosphatase provided removes phosphate in a quantitative manner allowing for accurate kinetic analysis

Amenable to High-throughput Analysis

• The assay can be performed in standard multi-well microplates
• Utilizes a standard colorimetric plate reader for analysis

Kit Contents

• CHO cell-expressed Recombinant Mouse CD39L2/ENTPD6
• ATP
• ADP
• Phosphatase Buffer 4
• Phosphate Standard
• Malachite Green Reagents A & B

Coupling Rates for Kinase Reactions

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.

• Coupling rates for 10 minute kinase reactions
• Coupling rates for 20 minute kinase reactions
• Coupling rates for 30 minute kinase reactions

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.

Data Example

Activity of PKA C beta

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.

Protein kinases are the largest class of enzymes in the human genome. These enzymes regulate almost all cellular processes by adding phosphate groups to proteins, thereby modifying the activity, localization, and overall function of their targets. Consequently, abnormal activity of kinases underlies many diseases including cancer.