cAMP Functional Assay Services

As an industry-leading CRO company focusing on GPCR ligand binding assay, Creative Biolabs provides multiple methods for cAMP functional assay to let our customers have flexible choices.

One of the prominent assay systems to study GPCR-mediated G_s activation is measurements of intracellular cAMP as an indirect method. Assays measuring cellular levels of cAMP are dependent on the activity of adenylyl cyclase, which is regulated by GPCRs coupled to Gα_s or Gα_i/o protein. Gα_s positively stimulate the activity of adenylate cyclase, resulting in increased cellular cAMP. In contrast, activation of Gα_i leads to negative regulation of adenylate cyclase and a decrease in cAMP levels.

Basic Methods of cAMP Functional Assay

cAMP levels are typically measured using a competition assay in which cellular cAMP competes with an introduced, labeled form of cAMP for binding to an anti-cAMP antibody. The principle is based on the competition between endogenous cAMP in samples or standard or calibrators, and horseradish peroxidase (HRP)-labeled cAMP in the kit for binding sites on the anti-cAMP antibodies. In the absence of cAMP, a majority of the HRP-cAMP conjugate is bound to the antibody, and increasing concentrations of cellular cAMP competitively displace the amount of bound conjugate, which results in reduced HRP activity and signal intensity. The assay is very flexible as it requires only a single washing step, reading can be from as little as 10min to 24 h following substrate addition, and can be used to measure adenylate cyclase activity or intracellular cAMP. The procedure can be modified for multiple agonists or antagonists (e.g., the beta-adrenergic agonist isoproterenol or antagonist propranolol).

Fig. 1. Schematic flowchart for the cAMP assay. (Vasudevan, 2017)

Bead-based proximity chemiluminescent assay

Cellular cAMP competes with a biotinylated cAMP probe recognized by a streptavidin donor and anti-cAMP antibody-conjugated acceptor beads. Release of the biotinylated cAMP from the antibody results in the dissociation of the streptavidin donor from its acceptor, which can be measured as a decrease in the chemiluminescent signal.

Fluorescence polarization (FP)-based cAMP Assay

When exposed to polarized light, the emission from an antibody-bound fluorescent-labeled cAMP is also polarized due to restricted molecular rotation. When the labeled cAMP is replaced on the antibody by cellular cAMP, the emission becomes more depolarized because it can rotate freely in solution. With the availability of red-shifted fluorophores, the signal-to-noise ratios have been greatly improved.

Our Feature Service

Support multiple assay methods
Experienced experts help optimize projects.
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Creative Biolabs is professional in GPCR ligand-binding assay and has experienced experts to help optimize the experiment scheme. Our multiple cAMP functional assays include but are not limited to Bead-based proximity chemiluminescent, FP-based assay. If you are interested in our services, please don't hesitate to contact us for more information.

Reference

Vasudevan, N. T. cAMP assays in GPCR drug discovery. Methods Cell Biol. 2017, 142: 51-57.

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