Screen Quest™ TR-FRET No Wash cAMP Assay Kit

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.

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.

Well	Volume	Reagent
CS1-CS7	25 µL	Serial Dilution
BL	25 µL	Diluent (Component E)
TS	25 µL	Test 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

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.
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.
Incubate the reaction at room temperature for 30 minutes.
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.
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.
Incubate the reaction at room temperature for 30 minutes.
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

Page updated on November 23, 2024

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