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Quantify cAMP by Absorbance or Fluorescence

Cyclic adenosine monophosphate (cAMP) is an important second messenger in many biological processes. cAMP is derived from adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway. cAMP is synthesized from ATP by adenylyl cyclase located on the inner side of the plasma membrane. Adenylyl cyclase is activated by a range of signaling molecules through the activation of adenylyl cyclase stimulatory G (Gs)-protein-coupled receptors and inhibited by agonists of adenylyl cyclase inhibitory G (Gi)-protein-coupled receptors. Liver adenylyl cyclase responds more strongly to glucagon, and muscle adenylyl cyclase responds more strongly to adrenaline.

 

Colorimetric ELISA cAMP Assay



cAMP dose response was measured with Screen Quest™ Colorimetric ELISA cAMP Assay Kit in a clear 96-well plate with a SpectraMax micro-plate reader (Molecular devices). As low as 0.1 nM cAMP can be detected in a 100 µL reaction volume at 405, 650 and 750 nm.
Screen Quest™ Colorimetric ELISA cAMP Assay kit is based on the competition between HRP-labeled cAMP and non-labeled cAMP. HRP-cAMP is displaced from the HRP-cAMP/anti-cAMP antibody complex by unlabeled free cAMP. In the absence of cAMP, HRP-cAMP conjugate is bound to anti-cAMP antibody exclusively. However, the unlabeled free cAMP in the test sample competes for anti-cAMP antibody with the HRP-cAMP antibody conjugate, therefore inhibits the binding of HRP-cAMP to anti-cAMP antibody.

Screen Quest™ Colorimetric ELISA cAMP Assay Kit provides a sensitive method for detecting adenylate cyclase activity in biochemical or cell-based assay system. Compared to other ELISA cAMP assay kits, our kit eliminates the tedious acetylation step. The kit uses Amplite® Green as a colorimetric substrate to quantify the HRP activity. The assay can be per-formed in a convenient 96-well plate.
 

Fluorimetric ELISA cAMP Assay



cAMP dose response was measured with Screen Quest™ Fluorimetric ELISA cAMP Assay Kit in a solid black 96-well plate with a Gemini microplate reader (Molecular Devices). As low as 0.1 nM cAMP was detected in a 100 µL reaction volume.
Screen Quest™ Fluorimetric ELISA cAMP Assay Kit provides a sensitive method for detecting adenylate cyclase activity. Compared to other commercial ELISA cAMP assay kits, this cAMP assay kit only requires a single wash step to remove unbound material prior to the development step. It also eliminates the tedious acetylation step. The kit uses Amplite® Red as a fluorogenic HRP substrate to quantify the HRP activity. The fluorescent product formed is proportional to the activity of HRP-cAMP conjugate.
 

Live Cell cAMP Assay



Screen Quest™ Live Cell cAMP Assay Principle.
Screen Quest™ Live Cell cAMP Assay Service Pack provides the real-time monitoring of intracellular cAMP change in a high-throughput format without a cell lysis step. The assay works through the cell lines that contain either an exogenous cyclic nucleotide-gated channel (CNGC) or the promiscuous G-protein, Gα16. The channel is activated by elevated levels of intracellular cAMP, resulting in ion flux and cell membrane depolarization which can be detected with a fluorescent calcium (such as Fluo-8® AM and Cal-520™ AM). Co-expression of Gα16with specific non-Gq-coupled receptors will result in the generation of an intracellular calcium signal upon receptor stimulation. The Screen Quest™ Live Cell cAMP Assay Service Pack provides both cell lines and reagents.
 

Table 1. Screen Quest™ cAMP Assay Kits

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
Price
36370Screen Quest™ Colorimetric ELISA cAMP Assay Kit650 1 plate$327
36371Screen Quest™ Colorimetric ELISA cAMP Assay Kit650 10 plates$2153
36373Screen Quest™ Fluorimetric ELISA cAMP Assay Kit5715851 plate$327
36374Screen Quest™ Fluorimetric ELISA cAMP Assay Kit57158510 plates$2153
36382Screen Quest™ Live Cell cAMP Assay Service Pack490520EachInquire