Product Background: The assay is a competitive ELISA in which the signal is inversely proportional to the amount of PIP3 detected. Once PIP3 has been extracted from millions of cell samples, it is first incubated with a PIP3 detector protein, then added to the PIP3-coated microplate for competitive binding. A peroxidase-linked secondary detection reagent and colorimetric substrate is used to detect the PIP3 detector protein bound to the plate. The colorimetric signal is inversely proportional to the amount of PIP3 extracted from cells.
The production of PIP3 from PI(4,5)P2 (PIP2) by class I PI3-kinases (PI3-K) is important in multiple cell signaling pathways. Typically, experiments to measure PI3-K activity have involved phosphorylation of a phosphoinositide substrate using 32P, then extraction of radioactive products, and separation using thin-layer chromatography. The assay plate method developed by Echelon Biosciences, Inc. allows the user to determine PI3-K activity by measuring the amount of PIP3 extracted from cells by means of standard ELISA format, eliminating the need for radioactivity, and thin layer chromatography.
Featured in Publications:
1. Ehrmantrout, Kimberly K., Branch, Angie M., Neilsen, Paul O., and Thompson, James E.."Nonradioactive Quantification of Phosphoinositide 3-Kinase (PI3K) Products from Breast Cancer Cell Lines". American Society for Cell Biology (ASCB), Washington, DC. 2007.
2. Ehrmantrout, Kimberly K., Branch, Angie M., Neilsen, Paul O.. "Quantification of PI3-Kinase Products from Breast Cancer Cell Lines". American Association for Cancer Research (AACR). Denver, CO. 2009.
3. King, JS., Teo, R., et al. “The Mood Stabilizer Lithium Suppresses PIP3 Signaling in Dictyostelium and Human Cells.” Disease Models and Mechanisms 2(5-6):306-12
4. Beneteau, M., Pizon, M., et al. “Localization og Fas/CD95 into the Lipid Rafts on Down-Modulation of the Phosphatidylinositol 3-Kinase Signaling Pathway.” Mol Cancer Res 6(4):604-613
5. Costa, C., Ebi, H., et al. "Measurement of PIP3 Levels Reveals an Unexpected Role for p110beta in Early Adaptive Responses to p110alpha-Specific Inhibitors in Luminal Breast Cancer" Cell 27(1):97-108
6. Gross, C., C.-W. Chang, et al. (2015). "Increased Expression of the PI3K Enhancer PIKE Mediates Deficits in Synaptic Plasticity and Behavior in Fragile X Syndrome." Cell Reports, DOI:10.1016/j.celrep.2015.03.060
7. S. Choi, et al. "Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases" Nature Cell Biology (2016) 18(12), 1324-35, doi:10.1038/ncb3441
Echelon's PIP Mass Assays
PI(4,5)P2 Mass ELISA
PI(3,4)P2 Mass ELISA
PI3 Kinase: PI3K alpha enzyme, active, PI3K Inhibitors, and PI3K Activity ELISA
Protein Kinases and Phosphatases
Keywords: Phosphatidylinositol (3,4,5)-trisphosphate, PtdIns(3,4,5)P3, PI(3,4,5)P3, PIP3, Phosphatidylinositol 3-kinase, PI 3-kinase, PI3-Kinase, PI3-K, PI3K, PI3 Kinase, PI3 K, cell extraction, lipid extraction