Cell Signaling Technology

Product Pathways - Screening Technologies

Cyclic AMP XP® Chemiluminescent Assay Kit #8019

The rabbit monoclonal antibody contained in this kit was developed using XMT®.

Kit IncludesQuantity
cAMP Ab Coated Microwells 96 tests
cAMP-HRP Conjugate 5.5 ml
cAMP Standard 0.5 ml
Luminol/Enhancer Solution 3 ml
Stable Peroxide Buffer 3 ml
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 10 ml
Cell Lysis Buffer (10X) #9803 15 ml

Species Cross-Reactivity

All

Reactivity Key: All=All species expected
Species cross-reactivity is determined by western blot.

Description

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.

Specificity / Sensitivity

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).

ELISA

ELISA

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.

ELISA

ELISA

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 emissionat 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.

Background

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).

  1. Serezani, C.H. et al. (2008) Am J Respir Cell Mol Biol 39, 127-32.
  2. Beavo, J.A. and Brunton, L.L. (2002) Nat Rev Mol Cell Biol 3, 710-8.
  3. Kopperud, R. et al. (2003) FEBS Lett 546, 121-6.
  4. Kamenetsky, M. et al. (2006) J Mol Biol 362, 623-39.
  5. Cheng, J. and Grande, J.P. (2007) Exp Biol Med (Maywood) 232, 38-51.
  6. Holz, G.G. et al. (2006) J Physiol 577, 5-15.
  7. Taylor, S.S. et al. (2008) Biochim Biophys Acta 1784, 16-26.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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