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Enzyme Immuno Assay Protocol

A. Reagent Preparation

  1. Bring all microwell strips to room temperature before use.
  2. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each kit) in Milli-Q or equivalently purified water.
  3. Dilute the 10X Cell Lysis Buffer #9803 to 1X in Milli-Q or equivalently purified water. 1 mM phenylmethylsulfonyl fluoride (PMSF) should be added fresh each time. This buffer can be stored at 4°C for short-term use (1–2 weeks).

B. Cell Lysate Preparation

NOTE: This procedure is for a 96-well tissue culture plate. It can be modified for other tissue culture plates (6, 12, 24, or 48 well).

  1. Plate cells of interest in 96-well plate (typically between 6–100 X 103 cells/well) and incubate overnight under appropriate cell culture conditions.
  2. Rinse cells with 200 μl warm PBS, then add test compounds in serum free mediums and incubate cells for the desired time period.
  3. Rinse cells twice with 200 µl ice cold PBS, and then add 100 μl/well 1X lysis buffer, keep cells on ice for 5 to 10 minutes.

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.

C. Assay

  1. Bring all kit components to room temperature.
  2. Add 50 µl of the HRP-linked target solution and 50 μl sample to the antibody-coated assay plate. Cover the plate and incubate at room temperature for 3 hours on a horizontal orbital plate shaker.
  3. Discard plate contents and wash wells 4 times with 200 μl /well of 1X Wash Buffer. Make sure to discard all liquid after each wash but do not allow wells to completely dry.
  4. Add 100 μl TMB substrate.
  5. Incubate for 30 minutes at room temperature.

NOTE: Watch the color as it being developed since it may be necessary to stop the reaction before 30 minutes.

  1. Add 100 μl STOP solution.
  2. Measure absorbance at 450 nm (for optimal results, read the plate within 30 minutes after adding STOP solution).

NOTE: To determine the absolute amount of the tested substance, a standard curve needs to be generated each time. Please follow the detailed protocol on the product datasheet to determine the concentration range for the standard curve.

posted April 2010

protocol id: 68

Product Includes Quantity (with Count) Solution Color
cAMP Ab Coated Microwells 96 assays
cAMP-HRP Conjugate 11 ml Red
cAMP Standard (2.4uM) 1 ml Colorless
TMB Substrate 7004 11 ml Colorless
STOP Solution 7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 10 ml Colorless
Cell Lysis Buffer (10X) 9803 15 ml Yellowish

Product 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).


Species Reactivity: 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).


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.



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