Cell Signaling Technology

Product Pathways - Screening Technologies

Cyclic AMP XP® Assay Kit #4339

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

Kit IncludesQuantity
cAMP Ab Coated Microwells 96 assays
cAMP-HRP Conjugate 11 ml
cAMP Standard 1 ml
TMB Substrate #7004 11 ml
STOP Solution #7002 11 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® 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 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, HRP substrate TMB is added to develop color. Because of the competitive nature of this assay, the magnitude of the absorbance for this developed color is inversely proportional to the quantity of sample cAMP. Measurement of absorbance using the cAMP Standard allows calculating the absolute amount of cAMP in a sample of interest.Note: 12 8-well modules -Each module is designed to break apart for 8 tests.

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 treatments are shown in Figure 2 (CHO cells) and Figure 3 (293 cells).

Standard curve

Standard curve

Figure 1: cAMP Standard was diluted in 1X Cell Lysis Buffer #9803 and samples were assayed following the Cyclic AMP XP® 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.

Cell-based assay

Cell-based assay

Figure 2: Treatment of CHO cells with Forskolin #3828 increases cAMP concentration as detected by Cyclic AMP XP® Assay Kit #4339. CHO cells were seeded at 4x104 cells/well in a 96-well plate and incubated overnight. Cells were either left untreated or pretreated with 0.5 mM IBMX for 30 minutes prior to forskolin treatment (15 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(A-Abasal)/(Amax-Abasal)], where A is the sample absorbance, Amax is the absorbance at maximum stimulation (i.e., high forskolin concentration), and Abasal is the absorbance 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.

Cell-based assay

Cell-based assay

Figure 3: Treatment of 293 cells with isoproterenol increases the cAMP concentration as detected by Cyclic AMP XP® Assay Kit #4339. 293 cells were seeded at 3x104 cells/well in a 96-well plate and incubated overnight. Cells were pretreated with 0.5 mM IBMX for 30 minutes prior to isoproterenol treatment (3 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follow: % activity=100*[(A-Abasal)/(Amax-Abasal)], where A is the absorbance of the sample, Amax is the absorbance at maximum stimulation (i.e., high isoproterenol concentration), and Abasal is the absorbance at basal level (no isoproterenol). Isopropterenol is a β-adrenoceptor agonist and activates beta-2 adrenergic receptors (ADRB2) that are endogenously expressed on 293 cells. Activation of ADRB2 then leads to activation of adenylyl cyclase and synthesis of cAMP as its second messenger.


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

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

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

Products