UniOne® TR-FRET Human cAMP Hirange Detection Kit

The kit employs homogeneous time-resolved fluorescence technology (TR-FRET) to precisely measure cAMP produced by cells. This method can rapidly detect changes in cAMP accumulation upon inhibition or activation of Gs- or Gi-mediated signaling pathways.

As shown in the figure, the Eu-labeled anti-cAMP antibody (TR-FRET donor) and the Ac-labeled cAMP (TR-FRET acceptor) bind directly, bringing the donor and acceptor antibodies into close proximity. Excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific emission signal at 665 nm. When cAMP is present in cell samples or standard curves, it competes with the Ac-labeled cAMP for binding to the Eu-labeled anti-cAMP antibody, thereby reducing the FRET signal. This specific signal is proportional to the extent of interaction between the Eu-labeled anti-cAMP antibody and the Ac-labeled cAMP. The homogeneous assay is easy to perform and requires no washing steps.

Caution: After the first thaw, immediately aliquot and store at the recommended temperature. Avoid storing diluted samples or subjecting them to repeated freeze-thaw cycles.

1. Reagent Preparation

1.1 Before use, thaw all reagents at room temperature (allow them to equilibrate at room temperature for at least 30 minutes). The reaction system in a 384-well plate is 20μL (the reagent volumes for the reaction system are shown in the table below). Calculate the required volume for your experiment before preparing, and prepare only as needed; the following preparation is for reference only, using 1,000 reactions as an example.

Table 1. Reagent Preparation

1.2 Gradient Dilution of Samples to Be Measured

Please prepare according to your experimental conditions; the following information is for reference only.

Taking cAMPcalibratoras an example, the diluent is Stimulation Buffer,andAdjust the sample to be tested according to its actual concentration.

Table 2.cAMPcalibratorconfigurationtable(adjust as needed)

1.3 Preliminary Processing Based on Cell Experiments

①Stimulation Buffer is used to dilute cells and stimulate drugs; the Stimulation Buffer provided in the kit is a solution without IBMX added. Depending on the experimental purpose, add an appropriate concentration of IBMX to prevent cAMP degradation.

②Before conducting the formal experiment, it is necessary to explore, based on the characteristics of the cells and the research drug, the number of cells added per well and the incubation time between cells and the drug, ensuring that the cAMP level produced by the cells falls within the linear range of the calibration curve.

2. Sample Addition and Controls

2.1 cAMP Standard Curve: Add 5 μL of gradient-diluted cAMP calibrator, 5 μL of Stimulation Buffer, and 5 μL of working solution of Anti-cAMP Eu antibody. After incubating for 10 minutes, add 5 μL of cAMP-Ac.

2.2 Test samples: Add 5 μL of gradient-diluted test samples, 5 μL of Stimulation Buffer, and 5 μL of working solution of Anti-cAMP Eu antibody. After incubating for 10 minutes, add 5 μL of cAMP-Ac.

2.3 Cell-based assay: Based on preliminary experiments, determine the cell concentration to be added; add 5 μL of cells and 5 μL of gradient-diluted drug to each well. Following the drug stimulation time established in the pre-experiment, incubate, then add 5 μL of working solution of Anti-cAMP Eu antibody, incubate at room temperature for 10 minutes, and subsequently add 5 μL of cAMP-Ac.

2.4 Negative control wells (NC): Replace the test sample with 5 μL of Stimulation Buffer, or use cells that have not been treated with the drug..

After all samples have been added, centrifuge, cover with a sealing film, and incubate at room temperature for 2 hours.

•The incubation time between cells and drugs needs to be explored independently; subsequent reagents should be added after stimulating the cells with the drug.

3.Detection

Detect on a microplate reader compatible with TR-FRET. Use an excitation wavelength of 320/340 nm, and measure emission wavelengths at 620 nm and 665 nm.

[Result Calculation]

1) Calculate the signal ratio: Divide the 665 nm fluorescence signal by the 620 nm fluorescence signal, then multiply the result by 10,000.

Ratio = (665/620)times10000

2) Calculate based on the signal valueNet signal:

Net signal= (Std-NC)/NC×100

3) Calculate the CV (%):

CV (%) = Standard Deviation/Mean Ratio × 100%

[Data Example]

The following data are for illustrative purposes only and should not be used in place of experimental results; outcomes may vary depending on the plate reader.It varies from device to device. 

1. cAMP Reference Standard Data

2. Evaluation of the cAMP matrix effect

3. Evaluation of cAMP Freeze-Thaw Stability

Note: RecommendedMicroplate (384-well plate, white, flat-bottom wells)