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Second Messenger Assays From DiscoveRx

Ligand induced GPCR modulation can be studied by measuring second messenger signaling molecules. G-protein coupled receptor (GPCR) coupled to Gi and Gs activates the cAMP pathway, while GPCR coupled to Gq activates Calcium. Second messenger assays allow the detection and characterization of all compound classes, full and partial agonist or antagonist, inverse agonist, and allosteric modulators. DiscoveRx offers a full range of cell lines and detection kits for the quantification of these events.

HitHunter® cAMP Assay Platform

Our HitHunter® cAMP assays are competitive immunoassays in which cAMP is labeled with β-galactosidase (β-gal) enzyme donor (ED).  This cAMP-ED conjugate and cAMP released from the testing cells compete for binding to an anti-cAMP antibody.  In the absence of the cellular cAMP, most of the cAMP-ED conjugate binds to the cAMP antibody, making them unable to complement with the enzyme acceptor (EA).  In the presence of cellular cAMP, the cAMP-ED is sequestered from the antibody by the cellular cAMP, so that it complements with EA to form active β-gal enzyme.  β-gal activity can be detected by DiscoveRx detection reagents using a chemiluminescent readout.

Features & Benefits

  • Works on any luminometer or multi-format reader
  • Unmatched performance for Gi-coupled receptors
  • Directly measure cAMP in cell-based assays- adherent & suspension mode
  • Ideal for cAMP-dependent phosphodiesterases
  • Chemiluminescent detection - minimizes false positives from autofluorescence

HitHunter® Calcium Assay Platform

DiscoveRx’s HitHunter® Calcium No WashPLUS kits use a homogenous assay format to detect calcium mobilization. Measurement of intracellular calcium provides valuable information on the activation status of GPCRs and ion channels. Our calcium assay kits provide a fast, reliable, low-cost alternative to traditional wash and quench-based assays.

Features & Benefits

  • Single step, no wash protocol
  • Large assay windows, larger dynamic range and  improved signal & performance over FLUO-3
  • Flexible protocol - use with adherent or non-adherent cells, GPCRs & calcium channels
  • Fewer false positives, due to reduced spontaneous calcium flux and minimal perturbation of cells
  • Validated for use with all Gq-coupled PathHunter® cell lines