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Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Published on: October 23, 2019
Development of a microsphere-based p38alpha MAP kinase no-wash assay
Stefan Laufer1, Sabine Linsenmaier
1Eberhard-Karls-University of Tübingen, Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Auf Der Morgenstelle 8 D-72076 Tübingen, Germany. Stefan.Laufer@Uni-Tuebingen.De
Journal of Biomolecular Screening
|June 9, 2006
Summary
A novel assay measures p38alpha mitogen-activated protein (MAP) kinase activity using microspheres. This nonradioactive, no-wash method simplifies drug candidate screening for p38alpha MAP kinase inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- p38alpha mitogen-activated protein (MAP) kinase is a key signaling molecule involved in cellular responses.
- Accurate measurement of p38alpha MAP kinase activity is crucial for drug discovery.
- Existing assays can be complex or require radioactive reagents.
Purpose of the Study:
- To establish a nonradioactive, microsphere-based, no-wash assay for measuring p38alpha MAP kinase activity.
- To optimize the assay conditions for reliable and efficient measurement.
- To validate the assay's performance by comparing results with known assays.
Main Methods:
- Utilized glutathione-S-transferase activating transcription factor 2 (GST-ATF-2) fusion protein as a substrate.
- Immobilized GST-ATF-2 onto glutathione-microspheres (GSH-microspheres).
- Measured substrate phosphorylation using a bi-phospho-specific antibody in the presence of p38alpha MAP kinase and test compounds.
Main Results:
- Successfully established and optimized a microsphere-based, no-wash assay for p38alpha MAP kinase activity.
- Demonstrated the assay's ability to measure kinase activity and evaluate inhibitors.
- Obtained IC50 values for p38alpha MAP kinase inhibitors comparable to those from established assays.
Conclusions:
- The developed assay provides a robust and convenient method for assessing p38alpha MAP kinase activity.
- This assay is suitable for high-throughput screening of drug candidates targeting p38alpha MAP kinase.
- The assay's nonradioactive and no-wash format offers advantages in simplicity and safety.
