Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction Universal Kinase Assay
2011
New Method for Measuring Kinase Activity Using CD39L2
publication
Evidence: high
Author Information
Author(s): Zhengliang L. Wu
Primary Institution: R&D Systems Inc.
Hypothesis
Can a new ADP-based phosphatase-coupled kinase assay improve the measurement of kinase activity?
Conclusion
The CD39L2-coupled kinase assay provides a simple, direct, and quantitative method for measuring kinase activity.
Supporting Evidence
- The assay achieved Z' factors greater than 0.5 for three different kinases, indicating high-throughput compatibility.
- CD39L2 showed a more than 50-fold preference for ADP over ATP, making it suitable for the assay.
- The method allows for the determination of kinetic parameters without completing the coupling reaction.
Takeaway
This study introduces a new way to measure how active certain proteins are by using a special enzyme that helps detect a product made during the reaction.
Methodology
The assay uses CD39L2 to hydrolyze ADP to AMP and phosphate, which is then detected using malachite green reagents.
Limitations
The assay's sensitivity is limited by the phosphate detection reagents, which have a lower limit for accurate detection.
Digital Object Identifier (DOI)
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