G protein-coupled receptors (GPCRs) are a large protein family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses. There are two principal pathways: cAMP and phosphatidylinositol.
Phosphatidylinositol Accumulation Assay
Phosphatidylinositol Mass ELISA
The assay is a competitive ELISA in which the signal is inversely proportional to the amount of Phosphatidylinositol produced. Once Phosphatidylinositol has been extracted from cell samples, it is first incubated with a Phosphatidylinositol detector protein, then added to the Phosphatidylinositol-coated microplate for competitive binding. A peroxidase-linked secondary detection reagent and colorimetric substrate is used to detect the Phosphatidylinositol detector protein binding to the plate. The colorimetric signal is inversely proportional to the amount of Phosphatidylinositol extracted from cells.
PI(3)P Mass ELISA
The PI(3)P Mass ELISA Kit allows for the determination of PI3-K activity by quantifying the amount of PI(3)P found in cells. After non-radioactive lipid extraction, the PI(3)P samples obtained from millions of cells are detected on a competitive ELISA. The cellular PI(3)P samples are mixed and incubated with a PI(3)P Detector protein, then added to a PI(3)P-coated microplate for competitive binding. A peroxidase-linked secondary detector and colorimetric detection is used to detect the PI(3)P protein binding to the plate. The assay is sensitive to 1.5 pmol PI(3)P. The colorimetric signal is inversely proportional to the amount of PI(3)P obtained.
PI(4)P Mass Strip
The PI(4)P Mass Strip kit is designed to quantify PI(4)P obtained from cell extractions through a simple lipid-protein overlay experiment. After following a non-radioactive lipid extraction protocol, the PI(4)P samples obtained from millions of cells are spotted onto a nitrocellulose strip which has been pre-spotted with PI(4)P standards and PIPn controls. Each strip has space for spotting 8 unknown cell extraction samples.
PI(3,4)P2 Mass ELISA
The PI(3,4)P2 Mass ELISA measures the amount of PI(3,4)P2 extracted from cells by means of a competitive ELISA, (96 well format).
PI(4,5)P2 Mass ELISA
Phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P2, is a ubiquitous lipid shown to play a central role in a variety of cell functions including the PKB/Akt pathway leading to cell growth, motility, inflammation, and apoptosis. PI(4,5)P2 is a substrate for class I phosphoinositide 3-kinase (PI3-K) and the product of PTEN phosphatase activity.
PIP3 Mass ELISA
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. Our platform allows 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.
Our Ins(1,4,5)P3 Assay Platform is designed for use in protein pull-down experiments to identify and characterize inositol phosphate binding proteins and measure Ins(1,4,5)P3 accumulation. Applications include the isolation of InsPn-binding proteins present in a cell lysate or in a mixture of in vitro translated peptides, for separation by SDS-PAGE and further analysis.
PI3-Kinase Activity ELISA
PI3-K's are ubiquitously expressed lipid kinases which phosphorylate PIP2 at the 3'-hydroxyl of the inositol ring producing PIP3. PIP3 has shown to be an important lipid second messenger with key roles in fundamental cellular responses such as proliferation and survival. Due to these interesting biological roles, PI3-K and PIP3 have become attractive targets for research and drug development in many diseases including inflammation and cancer.
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