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Screen Quest™ Colorimetric ELISA cAMP Assay Kit

Adenosine 3’, 5’ cyclic monophosphate (cAMP) is an important second messenger in intracellular signal transduction. Monitoring levels of cAMP is one of the most common ways to screen for agonists and antagonists of GPCRs. Screen Quest™ Colorimetric ELISA cAMP Assay Kit is based on the competition between HRP-labeled cAMP and non-labeled cAMP. HRP-cAMP is displaced from the HRP-cAMP/anti-cAMP antibody complex by unlabeled free cAMP. In the absence of cAMP, HRP-cAMP conjugate is bound to anti-cAMP antibody exclusively. However, the unlabeled free cAMP in the test sample competes for anti-cAMP antibody with the HRP-cAMP antibody conjugate, therefore inhibits the binding of HRP-cAMP to anti-cAMP antibody. Our Screen Quest™ Colorimetric ELISA cAMP Assay Kit provides a sensitive method for detecting adenylate cyclase activity in biochemical or cell-based assay system. Compared to other ELISA cAMP assay kits, our kit eliminates the tedious acetylation step. The kit uses Amplite® Green as a colorimetric substrate to quantify the HRP activity. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Example protocol

AT A GLANCE

Protocol Summary
  1. Prepare samples
  2. Add 75 µL/well of cAMP standard or test samples into the anti-cAMP coated 96-well plate
  3. Incubate at room temperature for 5-10 mins
  4. Add 25 µL/well of 1X HRP-cAMP Conjugate
  5. Incubate at room temperature for 3 hours
  6. Wash 4 times with 200 µL/well Washing Buffer
  7. Add 100 µL/well of Amplite™ Green
  8. Incubate at room temperature for 1 to 3 hours
  9. Monitor absorbance increase at 405, 650 or 740 nm
Important Note

Do not freeze Anti-cAMP Ab Pre-coated 96-well plate (Component F), store it at 4°C. Allow all the kit components to warm to room temperature before using them. Some material might be stick to the vial cap during the shipment. Briefly centrifuge the vial to collect all the content.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

cAMP stock solution (100 µM)

Add 1 mL of Assay Buffer (Component B) to the vial of cAMP Standard (Component A).

HRP-cAMP conjugate stock solution (50X)

Add 55 µL (Cat. # 36370) or 550 µL (Cat. # 36371) of Assay Buffer (Component B) into the vial of HRP-cAMP Conjugate (Component C).
Note         The unused 50X HRP-cAMP conjugate stock solution should be divided into single use aliquots and stored them at -20 oC.

Washing solution (1X)

Add 1 mL of 10X Wash Solution (Component D) to 9 mL distilled water.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/36370

cAMP standard
Make 1:10, 1:100 and 1:3 serial dilutions of cAMP standards in Assay Buffer (Component B) to have 10,000, 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 nM cAMP diluted solutions. Store on ice or 4°C.

PREPARATION OF WORKING SOLUTION

HRP-cAMP Conjugate working solution

Make 1:50 dilution with Assay Buffer (Component B) to have 1X HRP-cAMP conjugate working solution before use. Store it on ice or 4°C.
Note         25 µL of 1X HRP-cAMP conjugate working solution is enough for one assay point; prepare appropriately volume for single use only.

    SAMPLE EXPERIMENTAL PROTOCOL

    Prepare samples
    1. Treat cells as desired:The following is an example of Hela cells treated with Forskolin to induce cAMP in a 96-well plate format: Aspirate off cell growth medium, add 100 µL/well 100 µM Forskolin in Hanks and 20 mM Hepes buffer (HHBS), incubate in a 5% CO2, 37°C incubator for 15 minutes. Aspirate off cell solution after the incubation, add 100 µL/well of Cell Lysis Buffer (Component E), and incubate at room temperature for another 10 minutes. This cell lysate can be assayed directly or after diluted in Assay Buffer (Component B).

      Note         Each cell line should be evaluated on an individual basis to determine the optimal cell density. Cells may be seeded the day before or on the day of the experiment depending upon the cell type and/or the effect of the test compounds.
    2. Tissue Samples:It is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen) due to quick metabolism of cyclic nucleotides in tissue. Weigh the frozen tissue and add 10 - 20 µL/mg of cell lysis buffer. Homogenize the sample on ice. Spin at top speed for 5 minutes and collect the supernatant. The supernatant may be assayed directly.
    3. Urine, Plasma and Culture Medium Samples:Urine and plasma may be tested directly with 1:200 to 1:1000 dilutions in 1X Lysis Buffer. Culture medium can also be tested with 1:10 to 1:200 dilutions in Lysis Buffer.

      Note         RPMI medium may contain > 350 fmol/µL cAMP.
    cAMP assay
    1. All the assay wells will be prepared in the following orders: A) cAMP standards, control, or tests samples; B) HRP-cAMP Conjugate.
    2. Add 75 µL/well of the cAMP diluted standard solution and test samples into each well of the anti-cAMP Ab coated 96-well plate (Component F). We recommended duplicating the assays for each standard and testing sample. Incubate at room temperature for 5 to 10 minutes.
    3. Add 25 µL/well of 1X HRP-cAMP Conjugate working solution. Incubate at room temperature for 3 hours by placing the plate on shaker.
    4. Aspirate plate contents, and wash 4 times with 200 µL/well of 1X wash solution.
    5. Add 100 µL/well of Amplite™ Green (Component G) into each well, and incubate at room temperature for 60 mins to 3 hours, protected from light.
    6. Monitor the absorbance increase at 405 nm, 650 nm, or 740 nm using an absorbance plate reader.

    Product family

    NameExcitation (nm)Emission (nm)
    Screen Quest™ Fluorimetric ELISA cAMP Assay Kit571584

    Citations

    View all 3 citations: Citation Explorer
    Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling
    Authors: Avanzato, D and Genova, T and Pla, A Fiorio and Bernardini, M and Bianco, S and Bussolati, B and Mancardi, D and Giraudo, E and Maione, F and Cassoni, P and others, undefined
    Journal: Scientific Reports (2016)
    The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice
    Authors: Tomankova, Hana and Valuskova, Paulina and Varejkova, Eva and Rotkova, Jana and Benes, Jan and Myslivecek, Jaromir
    Journal: Stress (2015)
    THE EFFECTS OF RESTRAINT STRESS ON HEART M
    Authors: Valu{\v{s}}kov{\'a}, Paulina and Tomankov{\'a}, Hana and Rotkov{\'a}, Jana and Va{\v{r}}ejkov{\'a}, Eva and Bene{\v{s}}, Jan

    References

    View all 132 references: Citation Explorer
    cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation
    Authors: Rodriguez P, Rojas J.
    Journal: J Cell Biochem (2016): 741
    Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism
    Authors: Alqurashi M, Gehring C, Marondedze C.
    Journal: Int J Mol Sci (2016): 852
    Role of the cAMP Pathway in Glucose and Lipid Metabolism
    Authors: Ravnskjaer K, Madiraju A, Montminy M.
    Journal: Handb Exp Pharmacol (2016): 29
    Odor-induced cAMP production in Drosophila melanogaster olfactory sensory neurons
    Authors: Miazzi F, Hansson BS, Wicher D.
    Journal: J Exp Biol (2016): 1798
    A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death
    Authors: Wang Z, Liu D, Varin A, Nicolas V, Courilleau D, Mateo P, Caubere C, Rouet P, Gomez AM, V and ecasteele G, Fischmeister R, Brenner C.
    Journal: Cell Death Dis (2016): e2198
    Page updated on December 6, 2024

    Ordering information

    Price
    Unit size
    1 plate
    10 plates
    Catalog Number
    3637036371
    Quantity
    Add to cart

    Additional ordering information

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    Storage, safety and handling

    H-phraseH303, H313, H333
    Hazard symbolXN
    Intended useResearch Use Only (RUO)
    R-phraseR20, R21, R22
    UNSPSC12352200

    Platform

    Absorbance microplate reader

    Absorbance405, 650, or 740 nm
    Recommended plateClear plate (Component F)

    Components

    cAMP dose response was measured with Screen Quest™ Colorimetric ELISA cAMP Assay Kit in a clear 96-well plate with a SpectraMax microplate reader. The Absorbance can be read at 405 nm (blue line), 650 nm (red line) or 740 nm (Green line), the data in figure B are from the incubation with Amplite® Green for 3 hours.
    cAMP dose response was measured with Screen Quest™ Colorimetric ELISA cAMP Assay Kit in a clear 96-well plate with a SpectraMax microplate reader. The Absorbance can be read at 405 nm (blue line), 650 nm (red line) or 740 nm (Green line), the data in figure B are from the incubation with Amplite® Green for 3 hours.
    cAMP dose response was measured with Screen Quest™ Colorimetric ELISA cAMP Assay Kit in a clear 96-well plate with a SpectraMax microplate reader. The Absorbance can be read at 405 nm (blue line), 650 nm (red line) or 740 nm (Green line), the data in figure B are from the incubation with Amplite® Green for 3 hours.
    cAMP dose response was measured with Screen Quest™ Colorimetric ELISA cAMP Assay Kit in a clear 96-well plate with a SpectraMax  microplate reader. A: The kit can detect as low as 0.1 nM cAMP in a 100 µL reaction volume at 405nm after incubation with Amplite® Green for 1 hour (blue line) and 3 hours (red line).
    Reduction of BTEC migration is dependent on [cAMP]<sub>i</sub> increase. (A) cAMP production upon stimulation with different purinergic agonists or CPA. ATP 100&thinsp;&mu;M and other antimigratory purinergic agonists induce a strong increase in intracellular cAMP level after 15&prime; of treatment, as shown in table A (data from at least three independent experiments are normalized to the control). (B) High cAMP levels are able to reduce BTEC migration in wound healing assay. Different inducers of intracellular cAMP increase were used: Forskolin 10 &mu;M (FK), 8-Br-cAMP 500 &mu;M and IBMX 100 &mu;M. Data are normalized on positive CNTRL at 8 hrs and expressed as mean&thinsp;&plusmn;&thinsp;S.E.M. Wilcoxon test: *p&thinsp;&lt;&thinsp;0.001 vs. CNTRL; &sect;p&thinsp;&lt;&thinsp;0.001 vs. ATP 100&thinsp;&mu;M; #p&thinsp;&lt;&thinsp;0.001. (C,i) Actin and paxillin staining in cells treated with FK 10&thinsp;&mu;M or not (CNTRL) for 5&prime;. Arrows in (C,i) indicate actin filaments distribution and phalloidin fluorescence intensity was evaluated along the major cell axis (white lines in the figures). Scale bar&thinsp;=&thinsp;10&thinsp;&mu;m (C,ii) Quantification of the cortical actin localization. (C,iii) Paxillin density quantification upon FK treatment. (C,iv) Total area measured for each condition. Data obtained from three independent experiments are expressed as mean&thinsp;&plusmn;&thinsp;S.E.M. Wilcoxon test: *p&thinsp;&lt;&thinsp;0.001 vs. CNTRL. (D) Effect on BTEC migration (wound healing) of different adenylyl cyclase modulators (ddAdo, NaHCO3), EPAC-1 activator (8-CPT) and PKA inhibitor (H89). Dotted line represents ATP-induced inhibition. Data are normalized on positive CNTRL at 8 hrs and expressed as mean&thinsp;&plusmn;&thinsp;S.E.M. Wilcoxon test: *p&thinsp;&lt;&thinsp;0.001 vs. CNTRL; &sect;p&thinsp;&lt;&thinsp;0.001 vs. ATP 100&thinsp;&mu;M; #p&thinsp;&lt;&thinsp;0.001. Source: <strong>Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells <em>via</em> cAMP signaling </strong>by Avanzato et al., <em>Scientific Reports</em>, Sept. 2016.