Figure 1: cAMP Standard was diluted in 1X Cell Lysis Buffer #9803 and samples were assayed following the Cyclic AMP XP®
Chemiluminescent Assay Kit protocol. This standard curve is for demonstration purposes only; users should generate a standard curve for each sample set in order to accurately determine cAMP concentration.
Figure 2: Treatment of CHO cells with Forskolin (FSK) #3828 increases cAMP concentration as detected by Cyclic AMP XP® Chemiluminescent Assay Kit #8019. CHO cells were seeded at 4x104 cells/well in a 96-well plate and incubated overnight. Cells were pretreated with 0.5 mM IBMX for 30 minutes prior to forskolin treatment (15 minutes) and lysed with 1X Cell Lysis Buffer #9803. The light emission values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(RLU-RLUbasal)/(RLUmax-RLUbasal)], where RLU is the sample relative light unit, RLUmax is the light emission at maximum stimulation (i.e., high forskolin concentration), and RLUbasal is the light emission
at basal level (no forskolin). Forskolin directly activates adenylyl cyclases and increases cellular cAMP concentration. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases and promotes accumulation of cAMP and cGMP in cells.
|Product Includes||Quantity (with Count)||Solution Color|
|cAMP Rabbit mAb Coated Microwells||1 x 96 tests|
|cAMP-HRP Conjugate||1 x 5.5 ml||Red|
|cAMP Standard (2.4 uM)||1 x 0.5 ml|
|Luminol/Enhancer Solution||1 x 3 ml|
|Stable Peroxide Buffer||1 x 3 ml|
|Sealing Tape||1 x 2 ea|
|ELISA Wash Buffer (20X) 9801||1 x 10 ml|
|Cell Lysis Buffer (10X) 9803||1 x 15 ml|
NOTE: If cell debris is observed it can be removed by brief centrifugation of the plate and transfer of the clear lysates to a new 96 well plate.
Make cAMP standard in the 1X Cell Lysis buffer: Take 50 µl of the cAMP standard (2.4 µM) and add it to 450 µl diluent to get 240 nM cAMP. Perform a 1:3 serial dilution of this standard to get 80 nM, 26.7 nM, 8.9 nM, 3.0 nM, 1.0 nM, 0.3 nM,and 0 nM. The diluent without cAMP will serve as the 0 nM cAMP.
NOTE: The standard curve is used to calculate the absolute amount of cAMP in the sample and is necessary for each assay.
Use a plate-based luminometer to measure Relative Light Units (RLU) at 425nM within 1-10 minutes following addition of the substrate. Optimal signal intensity is achieved when read within 10 minutes.
Protocol Id: 506
The Cyclic AMP XP® Chemiluminescent Assay Kit is a competition enzyme-linked immunoassay used to determine cAMP levels in cells or tissues of interest. In this assay, cAMP found in the test sample competes with a fixed amount of HRP-linked cAMP for binding to an anti-cAMP XP® Rabbit mAb immobilized onto a 96-well plate. Following washing to remove excess sample cAMP and HRP-linked cAMP, chemiluminescent reagent is added for signal development. Because of the competitive nature of this assay, the magnitude of light emission, measured in relative light units (RLU), is inversely proportional to the quantity of sample cAMP. Measurement of light emmision using the cAMP Standard allows calculating the absolute amount of cAMP in a sample of interest.
The immunoreactivity of this kit was tested against the following: ADP, AMP, ATP, cAMP, cGMP, cIMP, cTMP, CTP, GDP, GMP, and GTP. Relatively minor cross-reactivity was observed with cGMP and cIMP, with 10 fold higher sensitivity for cAMP compared to either cGMP or cIMP. No cross-reactivity was observed with any of the other factors tested. Kit sensitivity, as shown in Figure 1, demonstrates a dynamic range of 0.2 to 12 nM of cAMP. Changes in cellular cAMP levels following specific treatment with forskolin is shown in Figure 2 (CHO cells).
All Species Expected
Cyclic adenosine 3’,5’-monophosphate (cAMP) is an important second messenger involved in many signal transduction pathways in different cell types of numerous species (1-3). In mammalian cells, this important molecule is produced by adenylyl cyclases (AC). Extracellular stimuli such as neurotransmitters, hormones, chemokines, lipid mediators, and drugs can modulate AC activity to increase or decrease cAMP production by binding to a large number of transmembrane G protein-coupled receptors (4). The degradation of cAMP to AMP is catalyzed by phosphodiesterases that are regulated by intracellular nucleotide concentrations, phosphorylation, or binding of Ca2+/calmodulin and other regulatory proteins (5). A set of diverse molecules, including cAMP-dependent protein kinase (PKA), cyclic nucleotide-gated ion channels, and exchange proteins that are activated by cAMP (EPAC), mediate downstream cAMP signaling (6,7). cAMP modulates various biological processes including metabolism, differentiation, cardiac cell functions, neuronal signaling, cell adhesion, and immune functions (5-7).
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