HTRF IP-One Terbium-based assay performed on BMG LABTECH´s PHERAstar Plus

Franka Ganske, Laurence Jacquemart, Alexandre Jean, Marie-Laure Lebreton, Jean-Luc Tardieu

2008

BMG LABTECH, Germany. Cisbio Bioassays, France.

Inositol-1,4,5-triphosphate (IP3) is an important second messenger in the cell. It is synthesized during cascade reactions that happen after stimulation of a G-protein coupled receptor (GPCR). After activation of a G-protein coupled receptor phospholipase C (PLC) is released. This lipase cleaves the membrane lipid phosphatidylinosityl-bisphosphate (PIP2) into diacylglycerol and IP3. The main function of IP3 is to open calcium channels of the endoplasmic reticulum (ER) leading to the release of calcium ions into the cell.2 The possibility to determine the IP3 concentration would allow for studying potential activators or inhibitors of the G-protein coupled receptors. However, the IP3 molecule has extremely short half life, making it difficult to use as a measure of Gq receptor activation. It turned out that in presence of LiCl the downstream metabolite IP1 is stable and accumulates. Cisbio Bioassays developed highly accurate HTRF® assays for measuring IP1 in 96-, 384- and 1536-well formats providing HTS compatible assays. HTRF® (homogeneous time-resolved fluorescence) is based on a fluorescence resonance energy transfer between a Europium cryptate (donor) and a second fluorescent label (acceptor). Recently Cisbio Bioassays developed a new Terbium cryptate (Lumi4) that acts as a FRET donor. In this application note we show the comparison of BMG LABTECH’s PHERAstar and PHERAstar Plus microplate readers to measure the new Terbium-based IP-One Tb assay.

HTRF® Technology, GPCR research from A to Z , HTRF microplate readers

IP-One Gq assay kits