ScreenQuestFRET15002
ScreenQuestFRET8002

Call us today on 0800 724 633 or email info@scienze.nz to order and experience the AAT Bioquest Assay Advantage!

cAMP dose 

cAMP dose response was measured with Screen Quest™ FRET No WashcAMP Assay Kit using a ClarioStar microplate reader (BMG).

For more information please email info@scienze.nz  and one of our technical specialists will contact you directly to discuss your needs.

Screen Quest™ FRET No Wash cAMP Assay Kit

Overview

Screen Quest™ 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 fluorescent cAMP tracer and non-labeled free cAMP. Free cAMP displaces the fluorescent cAMP tracer from the HRP-cAMP/anti-cAMP antibody complex. The anti-cAMP antibody is labeled with our TR Fluor™ Eu while the cAMP tracer contains our trFluor™ 650. In the absence of cAMP, trFluor™ 650-cAMP conjugate is bound to TR Fluor™ Eu-labeled anti-cAMP antibody exclusively to have a strong FRET. However, the unlabeled free cAMP in the test sample competes for the TR Fluor™ Eu-labeled anti-cAMP antibody conjugate, 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 value due to the TR-FRET. The magnitude of FRET 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.

Platform

Fluorescence microplate reader
Recommended plate Solid black and/or Black wall/clear bottom
Instrument specification(s) Time-resolved

Components

Component A: Anti cAMP-trFluor™ Eu 1 vial
Component B: cAMP-trFluor™ 650 1 vial
Component C: cAMP Standard 1 vial (33 µg)
Component D: Cell Lysis Buffer 1 bottle (10 mL)
Component E: Diluent 1 bottle (10 mL)

Example protocol

Preparation of stock solution

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 -20oC.

Preparation of standard solution

For convenience, use our Serial Dilution Planner

Preparation of working solution

1. Anti cAMP-trFluor™ Eu working solution:
Add 55 µL (Cat. # 36379) or 550 µL (Cat. # 36380 or # 36381) of Diluent (Component E) into the vial of Anti cAMP-trFluor™ Eu (Component A). Add 50 µL of reconstituted solution 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 55 µL (Cat. # 36379) or 550 µL (Cat. # 36380 or # 36381) of Diluent (Component E) into the vial of cAMP-trFluor™ 650 (Component B). Add 50 µL of reconstituted solution to 2.5 mL of Cell Lysis Buffer (Component D). Note: Make soultion just before use and as per needed.

Procedure

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 oC 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.

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

 

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.

 

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 3. 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

Data Analysis

Results are Relative Fluorescence Units at 665nm and 620nm. Ratio is calculated as the F665nm / F620nm ratio and expressed in Δ F%.

R= F665nm/F620nm

Δ  F%= 100% x (R sample-R neg)/Rneg

Draw a standard curve by plotting Δ F% versus cAMP concentration.

Screen Quest™ FRET No Wash cAMP Assay Kit

Overview

Screen Quest™ 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 fluorescent cAMP tracer and non-labeled free cAMP. Free cAMP displaces the fluorescent cAMP tracer from the HRP-cAMP/anti-cAMP antibody complex. The anti-cAMP antibody is labeled with our TR Fluor™ Eu while the cAMP tracer contains our trFluor™ 650. In the absence of cAMP, trFluor™ 650-cAMP conjugate is bound to TR Fluor™ Eu-labeled anti-cAMP antibody exclusively to have a strong FRET. However, the unlabeled free cAMP in the test sample competes for the TR Fluor™ Eu-labeled anti-cAMP antibody conjugate, 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 value due to the TR-FRET. The magnitude of FRET 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.

Platform

Fluorescence microplate reader
Recommended plate Solid black and/or Black wall/clear bottom
Instrument specification(s) Time-resolved

Components

Component A: Anti cAMP-trFluor™ Eu 1 vial
Component B: cAMP-trFluor™ 650 1 vial
Component C: cAMP Standard 1 vial (33 µg)
Component D: Cell Lysis Buffer 1 bottle (10 mL)
Component E: Diluent 1 bottle (10 mL)

Example protocol

Preparation of stock solution

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 -20oC.

Preparation of standard solution

For convenience, use our Serial Dilution Planner

Preparation of working solution

1. Anti cAMP-trFluor™ Eu working solution:
Add 55 µL (Cat. # 36379) or 550 µL (Cat. # 36380 or # 36381) of Diluent (Component E) into the vial of Anti cAMP-trFluor™ Eu (Component A). Add 50 µL of reconstituted solution 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 55 µL (Cat. # 36379) or 550 µL (Cat. # 36380 or # 36381) of Diluent (Component E) into the vial of cAMP-trFluor™ 650 (Component B). Add 50 µL of reconstituted solution to 2.5 mL of Cell Lysis Buffer (Component D). Note: Make soultion just before use and as per needed.

Procedure

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 oC 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.

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

 

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.

 

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 3. 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

Data Analysis

Results are Relative Fluorescence Units at 665nm and 620nm. Ratio is calculated as the F665nm / F620nm ratio and expressed in Δ F%.

R= F665nm/F620nm

Δ  F%= 100% x (R sample-R neg)/Rneg

Draw a standard curve by plotting Δ F% versus cAMP concentration.

For more information please email info@scienze.nz  and one of our technical specialists will contact you directly to discuss your needs.

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