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Screen Quest™ TR-FRET No Wash cAMP Assay Kit

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Catalog Number36379
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Telephone1-408-733-1055
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Screen Quest™ TR-FRET No Wash cAMP Assay Kit provides a convenient assay method for monitoring the activation of adenylyl cyclase in G-protein coupled receptor systems. Compared to other commercial ELISA cAMP assay kits, this homogenous cAMP assay kit does not require a wash step or the acetylation step. The assay is based on the competition for a fixed number of anti-cAMP antibody binding sites between the trFluor™ 650 labeled cAMP tracer and non-labeled free cAMP. The anti-cAMP antibody is labeled with trFluor™ Eu while the cAMP tracer is labeled with trFluor™ 650. In the absence of cAMP, trFluor™ 650-cAMP conjugate is bound to trFluor™ Eu-labeled anti-cAMP antibody exclusively to have a strong FRET signal. While the unlabeled free cAMP is present in the test sample, it competes for the trFluor™ Eu-labeled anti-cAMP antibody conjugate binding sites, therefore inhibits the binding of trFluor™ 650-cAMP to anti-cAMP antibody. The trFluor™ 650 labeled cAMP tracer only has fluorescence lifetime of nanosecond while TR Fluor™ Eu-labeled anti-cAMP antibody-bound fluorescent cAMP tracer has much longer fluorescence lifetime of lanthanide fluorophore. The magnitude of time -resolved fluorescence signal (TR- FRET) signal is proportional to the concentration of cAMP in a sample. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format, and is convenient for monitoring the cAMP activity with ultra-specificity and sensitivity in G-protein coupled receptor systems.

Platform


Fluorescence microplate reader

Recommended plateSolid black and/or Black wall/clear bottom
Instrument specification(s)Time-resolved

Components


Component A: Anti cAMP-trFluor™ Eu1 vial
Component B: cAMP-trFluor™ 6501 vial
Component C: cAMP Standard1 vial (33 µg)
Component D: Cell Lysis Buffer1 bottle (10 mL)
Component E: Diluent1 bottle (10 mL)

Example protocol


CELL PREPARATION

For adherent cells
Plate cells overnight in growth medium at 30,000 -100,000 cells/well for a 96-well plate.

For non-adherent cells
Centrifuge the cells from the culture medium and then suspend the cell pellets in culture medium at 100,000-300,000 cells/well for a 96-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiment.

Treat cells as desired
The following is an example for Hela cells treated with Forskolin to induce cAMP in a 96-well plate format. 25µL cells in growth medium, add 25 µL/well 100 µM Forskolin in Hanks and 20 mM Hepes buffer (HHBS), incubate in a 5% CO2 , 37 °C incubator for 15 minutes.
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.

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 standard (1mM)
Add 100 µL Diluent (Component E) to cAMP Standard (Component C) and mix them well.
Note     The unused cAMP standard can be aliquoted and stored at -20 °C.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/36379


cAMP standard
1 mM stock soluton can be diluted to 11200 nM followed by 4X dilutions.

PREPARATION OF WORKING SOLUTION

1. Anti cAMP-trFluor™ Eu working solution
Add 50 µL of solution (Component A) to 2.5 mL of Cell Lysis Buffer (Component D).
Note     Make soultion just before use and as per needed.


2. cAMP-trFluor™ 650 working solution
Add 50 µL of solution (Component B) to 2.5 mL of Cell Lysis Buffer (Component D).
Note     Make soultion just before use and as per needed.

SAMPLE EXPERIMENTAL PROTOCOL


Table 1.Layout of cAMP standards and test samples in a solid black 96-well microplate. CS = cAMP standard (CS1-CS7); BL = blank control; TS = test sample.
BL BL TS TS
CS1 CS1 ... ...
CS2 CS2 ... ...
CS3 CS3    
CS4 CS4    
CS5 CS5    
CS6 CS6    
CS7 CS7    
Table 2. Reagent composition for each well.
WellVolumeReagent
CS1-CS725 µLSerial Dilution
BL25 µLDiluent (Component E)
TS25 µLTest Sample

Table 3.Overview of the protocol
cAMP Standard Cells
Negative Control Positive Control Standard Curve Negative Control Non-stimulated Stimulated
25 µL Diluent 25 µL Diluent 25 µL Standard 25 µL cells 25 µL cells 25 µL cells
25 µL  Compound Buffer 25 µL  Compound Buffer 25 µL Compound Buffer 25 µL Compound Buffer 25 µL Compound Buffer 25 µL Compound
Incubate 30 min at RT
25 µL Lysis Buffer 25 µL cAMP-trFluor™ 650 working solution 25 µL cAMP-trFluor™ 650 working solution 25 µL Lysis Buffer 25 µL cAMP-trFluor™ 650 working solution 25 µL cAMP-trFluor™ 650 working solution
25 µL Anti cAMP-trFluor™ Eu working solution
Incubate 30min at RT

Table 4.Compatible HTRF® plate readers
Manufacturers Instruments
Berthhold Technologies Tristar2 S; Mithras LB 940; Mithras2 LB 943
Hidex Sense; Sense Beta Plus
Molecular Devices Spectra Max i3X; Spectramax Paradigm; Spectramax M5e; Spectramax 3
Thermo Scientific Varioskan Lux
Biotek Synergy Neo2; Cytation 5; Cytation 3; Synergy H1; Synergy 2
BMG Labtech PHERAstar; CLARIOstar; POLARstar Omega; Fluostar Omega
Tecan Spark 10M; Infinite M100 Pro; Infinite F500; Infinite F200 Pro

cAMP assay in cell lysate
  1. Prepare and add cAMP standards (CS), blank controls (BL) and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 12.5 µL of each corresponding reagent instead of 25 µL.
    Note     Test samples could be Non-stimulated and/or stimulated samples.
  2. Add 25 µL of treatment (Compound resuspended in buffer) into each well of cAMP standard, blank control, and test samples to make the total cAMP assay volume of 50 µL/well. For a 384-well plate, add 12.5 µL of working solution into each well for a total volume of 25 µL/well.
  3. Incubate the reaction at room temperature for 30 minutes.
  4. Add 25 µL of cAMP-trFluor™ 650 working solution into each well of cAMP standard, blank control, and test samples to make the total cAMP assay volume of 75 µL/well. For a 384-well plate, add 12.5 µL of working solution into each well for a total volume of 37.5 µL/well.
    Note     For negative controls, Lysis Buffer can be added.
  5. Add 25 µL of cAMP-trFluor™ Eu working solution into each well of cAMP standard, blank control, and test samples to make the total cAMP assay volume of 100 µL/well. For a 384-well plate, add 12.5 µL of working solution into each well for a total volume of 50 µL.
  6. Incubate the reaction at room temperature for 30 minutes.
  7. Read on a compatible TR-FRET reader. 

Citations


View all 2 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)

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
The pleiotropic role of exchange protein directly activated by cAMP 1 (EPAC1) in cancer: implications for therapeutic intervention
Authors: Almahariq M, Mei FC, Cheng X.
Journal: Acta Biochim Biophys Sin (Shanghai) (2016): 75
A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors
Authors: Vedel L, Brauner-Osborne H, Mathiesen JM.
Journal: J Biomol Screen (2015): 849
Imaging alterations of cardiomyocyte cAMP microdomains in disease
Authors: Froese A, Nikolaev VO.
Journal: Front Pharmacol (2015): 172
Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2
Authors: Zoccarato A, Surdo NC, Aronsen JM, Fields LA, Mancuso L, Dodoni G, Stangherlin A, Livie C, Jiang H, Sin YY, Gesellchen F, Terrin A, Baillie GS, Nicklin SA, Graham D, Szabo-Fresnais N, Krall J, V and eput F, Movsesian M, Furlan L, Corsetti V, Hamilton G, Lefkimmiatis K, Sjaastad I, Zaccolo M.
Journal: Circ Res (2015): 707
cAMP controls the balance of the propulsive forces generated by the two flagella of Chlamydomonas
Authors: Saegusa Y, Yoshimura K.
Journal: Cytoskeleton (Hoboken) (2015): 412