Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format
Lipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we describe a method to prepare a DMSO-based lipid mixture that enables the 1536-well screening of the lipid kinase phosphatidylinositol-5-phosphate 4-kinase α (PI5P4Kα) utilizing the d-myo-di16-PtIns(5)P substrate in quantitative high-throughput screening (qHTS) format using the ADP-Glo™ technology to couple the production of ADP to a bioluminescent readout.
Key wordsQuantitative high-throughput screening (qHTS) PI5P4Kα Kinase Lipid Bioluminescence Luciferase ADP-Glo Phosphorylation
This work was supported by the Molecular Libraries Common Fund Program of the National Institutes of Health. The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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