| 货号 | 8019S |
| 描述 | 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. |
| 反应种属 | All |
| 应用 | ELISA |
| 目标/特异性 | 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). |
| 供应商 | CST |
| 背景 | 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). |
| 存放说明 | -20C |
| 参考文献 | 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. |
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. 图1：cAMP标准溶液用1X Cell Lysis Buffer #9803稀释，样本处理按照Cyclic AMP XP? Chemiluminescent Assay Kit指南操作。此标准曲线仅作为演示使用；使用者应为每一个样本组制作标准曲线，以备精确测定cAMP浓度。 | |
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. 图2：使用Forskolin(FSK) #3828处理CHO细胞，Cyclic AMP XP? Chemiluminescent Assay Kit #8019用来检测cAMP浓度的增加。CHO细胞种于4×10^4/孔的96孔板中孵育过夜。细胞0.5 mM IBMX处理30分钟后forskolin处理15分钟，然后用1X Cell Lysis Buffer #9803裂解。发光值（左）和活性百分数（右）如上所示。活性百分数按下述计算：% activity=100x[((RLU-RLUbasal)/(RLUmax-RLUbasal)]，RLU为样本发光度，RLUm是最大刺激（如高浓度forskolin）发光度，RLUbasal是基线水平（无forskolin）发光度。Forskolin直接激活腺苷环化酶，增加细胞内cAMP浓度。IBMX是非特异性cAMP和cGMP磷酸酶的抑制剂，可以促进cAMP和cGMP在细胞内积累。 |
