PIP3 Mass Assays

Features

• First non-radioactive PIP3 mass assays
  
• Quick, simple protocols
  
• Lipid protein overlay fomat
  
• ELISA plate format

Applications

• PIP3 quantitation from cell sample
  
• Determination of PI3K activity and its related growth signalling pathway

Description

The PIP3 Mass assays were designed to help researchers quantify PIP3 mass quickly and easily. They offer substantial time and cost savings when compared to radioactive methods. The production of PI(3,4,5)P₃ from PI(4,5)P₂ by type-1 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 PI(3,4,5)P₃ extracted from cells by means of standard ELISA format, eliminating the need for radioactivity, organic solvents, and thin layer chromatography.
The assay is a competitive ELISA in which the signal is inversely proportional to the amount of PI(3,4,5)P₃ produced. Once PI(3,4,5)P₃ has been extracted from cells samples, it is first incubated with a PI(3,4,5)P₃ detector protein, then added to the PI(3,4,5)P3-coated microplate for competitive binding. A peroxidase-linked secondary detection reagent and colorimetric substrate is used to detect PI(3,4,5)P₃ detector protein binding to the plate. The colorimetric signal is inversely proportional to the amount of PI(3,4,5)P₃ extracted from cells. The assay is sensitive to 1 pmol PIP3, and requires approximately ~3 x 10⁶ cells per data point depending on the experimental system used.
The PIP3 Mass Strip kit is designed to quantify PIP3 obtained from cell extractions or PI3K reactions through a simple lipid-protein overlay experiment.