Application note: Functional GPCR studies using AlphaScreen cAMP Detection Kit
Posted: 5 January 2018 | Dawn Nida (PerkinElmer), Jen Carlstrom (PerkinElmer) | No comments yet
In this application note, PerkinElmer demonstrates the ease and utility of the AlphaScreen® cAMP assay for detecting both agonist and antagonist-induced cAMP responses within cells.
G protein-coupled receptors (GPCRs) are cell surface transmembrane receptors that catalyze the activation of G-proteins. GPCRs are involved in a wide variety of physiological processes and represent an important class of pharmaceutical drug targets.
GPCR activity is commonly assessed by measuring levels of the intracellular cAMP levels upon stimulation by agonists and therefore, methods for detecting and quantifying cAMP are in high demand. There are a large number of assays available on the market to detect and quantify cAMP levels in cells. However, the ideal assay is a homogenous, non-radioactive assay that allows for sensitive and reproducible detection of cAMP.
In the AlphaScreen cAMP assay, a biotinylated-cAMP tracer molecule is captured by Streptavidin Donor beads and an anti-cAMP monoclonal antibody is conjugated to the AlphaScreen Acceptor beads. When the anti-cAMP antibody binds to the biotinylated cAMP, the Donor and Acceptor beads are brought into close proximity. Upon illumination at 680 nm, the Donor beads convert ambient oxygen to singlet oxygen, which can diffuse approximately 200 nm in solution. If an Acceptor bead is within that distance, energy is transferred to the Acceptor bead, resulting in light production between 520 and 620 nm.
Here we show that the AlphaScreen cAMP technology provides comparable assay pharmacology with expected rank order of agonist or antagonist potency in a cell-based, homogenous no-wash assay format. We also show that the AlphaScreen cAMP assay is a robust assay and suitable for screening.
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Assays, Cell-based assays, Drug Discovery Processes, GPCRs, Protein, Screening