Description
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 CD 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