GPCR Technology – Second Messenger
DiscoveRx offers a full range of functional assays designed to detect second messenger signaling molecules including cAMP and intracellular calcium. These assays enable detection and characterization of all compound classes - full and partial agonist, antagonist, inverse agonist, and allosteric modulators – providing robust and reliable detection and excellent miniaturization capabilities.
Naturally Coupled Gs, Gi & Gq Cell Lines
Over 130 naturally coupled clonal cell lines expressing Gs, Gi, or Gq coupled receptors that have been characterized for appropriate GPCR pharmacology, specificity and stability in culture. Assay protocols have been optimized for detection of cAMP (Gs and Gi) using HitHunter® cAMP assays and calcium flux (Gq) using the fluorescent based Calcium No WashPLUS reagent.
HitHunter® cAMP Assays
HitHunter® cAMP assays are competitive immunoassays competitive immunoassays in which the analyte e.g. cAMP is labeled with the β-galactosidase enzyme donor in a configuration that enables it to be detected by an anti-cAMP antibody. In the absence of cellular cAMP, the conjugated cAMP is sequestered by the antibody leaving the enzyme in its inactive state. In the presence of cellular cAMP, the enzyme donor conjugates are able to complement with the enzyme acceptor. β-gal activity can be detected using substrates that are converted to a chemiluminescent product.
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| Free cAMP from cell lysates competes for antibody binding against labeled cAMP (ED-cAMP conjugate). Unbound ED-cAMP is free to complement EA to form active enzyme by EFC, which subsequently hydrolyzes substrate to produce signal. A positive signal generated is directly proportional to the amount of free cAMP bound by the binding protein. |
The HitHunter® Advantage:
- Large assay windows– ideal for difficult Gi- antagonist screens
- Chemiluminescent detection - minimizes false positives from autofluorescence
- High serum tolerance - ideal for biological samples and large molecule therapeutics
- Broad dynamic range and high sensitivity - enables cAMP detection in primary cells and membrane preparations
- Versatile detection - perfect for all compound types (agonist, antagonist, partial or inverse agonist, allosteric modulator)
Calcium Flux Measurements
DiscoveRx offers naturally Gq-coupled GPCR assays that measure Calcium flux of the activated receptor in a live cell, non-imaging assay format. In this assay, a calcium sensitive dye is loaded into cells to allow for the real time detection of intracellular calcium mobilization. Activation of a GPCR by injection of a compound results in the release of calcium from intracellular stores and an increase in dye fluorescence that is measured in real time.
The Calcium NoWashPLUS Advantage
- No wash, one-step protocol
- Large assay windows - larger dynamic range, improved signal and better performance than FLUO-3
- Flexible protocol – use with adherent or non-adherent cells, GPCRs and calcium channels
- Fewer false positives – reduced spontaneous calcium flux and minimal perturbation of cells
- Validated for use with all Gq-coupled PathHunter® cell lines.