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HTRF IP-One Assay Performed on the PHERAstar and RUBYstar Plate Readers

Marjan Orban1, Francois Degorce2 and Jean-Luc Tardieu2
1BMG LABTECH, Germany; 2Cisbio international, France


Application Note 137 Rev. 12/2005

  • HTRF IP-One assay for Gq pathway investigation under HTS conditions
  • Comparable performance of PHERAstar and RUBYstar with the HTRF IP-One assay in terms of EC50 and Z' (> 0.78)
  • Comprehensive list of GPCRs already validated with IP-One assay for agonist responses
    •


    Introduction
    HTRF (homogeneous time-resolved fluorescence) technology, developed by Cisbio international, is used in assay development and drug screening. HTRF is based on FRET between a Eu3+ cryptate (donor) and a second fluorescent label (acceptor). A new acceptor, d2, allows the introduction of a complete GPCR (G protein-coupled receptor) platform suitable for drug discovery.

    Upon activation, GPCRs carry the information within the cell via two major signalling pathways: the activation of Gαs or Gαi coupled GPCRs results in a variation of the cAMP level, whereas the activation of Gq coupled GPCRs result in a transient increase of intracellular Ca2+ triggered by inositol (1,4,5) tri-phosphate (IP3). Cyclic AMP and IP3 therefore represent two essential secondary messengers for monitoring the activity of most GPCRs.

    Concerning the Gq pathway, the precursor molecule for the signaling cascade, IP3, is an extremely instable product (turnover only a few tens of seconds) and its degradat
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