HTRF IP-One assay performed on the PHERAstar FS microplate reader
G-protein coupled receptors (GPCR) convert external stimuli such as hormones and growth factors into intracellular actions. In case of Gq receptor stimulation, phospholipase C (PLC) is activated leading to cytoplasmic Ca2+ release and degradation of D-myo-inositol 1,4,5-triphosphate (IP3) to IP1.
The IP-One homogenous TR-FRET (HTRF®) assay by Cisbio Bioassays measures Gq activation by quantifying IP1. An IP1-specific antibody is coupled to the europium cryptate donor fluorophore. It binds to IP1 which is coupled to the acceptor fluorophore d2 leading to a high TR-FRET signal. However, if non-labelled IP1 in the sample cell lysate competes with the fluorophore-labelled IP1, HTRF signal decreases.
The PHERAstar multi-mode plate reader detected TR-FRET-changes in various cell lines expressing GPCR targets upon against addition. It provided robust assay readouts as indicated by Z' values >0.78 and EC50 values that match values reported in literature.