Thursday, November 29, 2012

Applications Thursday – GPCR Activation via cAMP or Inositol Phosphate Production is Measured in Live Cells Using HTRF® Chemistry

GPCRs have two major signaling pathways - the regulation of cAMP levels and the increase in intracellular Ca2+ triggered by inositol 1, 4, 5- triphosphate (IP3). Specific G proteins activates these signaling pathways and they are associated with different receptors. Activation of a Gs or Gi coupled receptor results in the increase or decrease of cAMP levels, respectively. While activation of a Gq coupled receptor activates phospholipase C (PLC) and triggers the inositol phosphate (IP) cascade.

Cisbio Bioassays has assay kits able to measure the activation of Gs, Gi and Gq coupled receptors in one assay, using their new generation Lumi4-Tb™ TR-FRET Cryptate. This chemistry allows for the detection of two signals in one well using a green acceptor (λ = 520 nm) and a red acceptor (λ = 665 nm). This same chemistry is used in Cisbio’s Tag-lite® technology.

IP-One and cAMP experiments were performed on the PHERAstar HTS microplate readers using Simultaneous Dual Emission detection (detailed in Application note 209 . With this unique feature, the plate is read only once for dual emission assays, thereby decreasing time and variability. All Cisbio chemistry can be performed on the PHERAstar FS, which uses a UV Laser for excitation. The UV Laser will allow for greater sensitivity and faster read times (see Application Note 222 for IP-One data using the UV Laser) . The POLARstar and FLUOstar Omega with advanced assay technology also perform this chemistry, but in a non-HTS fashion.