UA-Glo® Firefly/Nano Dual Luciferase Assay System

The dual-luciferase reporter gene system is widely used in mammalian cell function research, including cell metabolism, signaling pathways, transcription factors, gene function, and drug screening. In the dual-luciferase reporter system, one luciferase serves as the experimental reporter gene to indicate the activity of the target under study, while the other luciferase acts as the control reporter gene to normalize the activity of the experimental reporter gene. This minimizes experimental errors caused by factors such as transfection efficiency, cell count, cell lysis effects, and sample volume.

The UA-Glo® Firefly/Nano Dual-Luciferase Assay Kit enables the detection of Firefly luciferase and Nano-Luc (Nano-Luciferase) expression in the same sample, offering advantages such as high sensitivity, excellent stability, reproducibility, and ease of use. Compared to traditional Firefly/Renilla luciferase dual-luciferase detection systems, the UA-Glo® Firefly/Nano Dual-Luciferase Assay Kit provides higher signal values, greater sensitivity, and improved tolerance to compound effects on luciferase, significantly reducing false-positive results in high-throughput screening due to compound interference with luciferase activity.

The Firefly/Nano Dual Luciferase Assay Kit consists of R1 reagent, R2 buffer, and R2 substrate. The R1 reagent is filled in brown bottles, the R2 buffer is filled in white bottles, and the R2 substrate is aliquoted in microtubes. The specifications are as follows:

Specification	R1 Reagent	R2 Buffer	R2 Substrate	Detectable 96-well Plate Wells	Detectable 384-well Plate Wells
10 ml	10 ml	10 ml	100μl	200	1,000
100 ml	100 ml	100 ml	1ml	2,000	10,000
10x100 ml	10x100 ml	10x100 ml	10x1ml	2,000	100,000

 

1.Seed cells at an appropriate density in 96-well or 384-well white cell culture plates. Transfect the cells with vectors expressing firefly luciferase and Nano-Luc luciferase as required by the experiment, or directly use stable cell lines expressing these dual luciferases.

2.Treat the cells according to experimental requirements and culture for an appropriate duration.

3.Remove the cell culture plate and equilibrate it to room temperature (22-25°C) [Note 1].

4.Remove the detection reagents and equilibrate them to room temperature. R1 reagent can be used directly. R2 substrate is a 100x concentrated solution and should be diluted with R2 buffer to prepare a 1x R2 working solution before use [Note 2].

5.Add an equal volume of R1 reagent to the cell culture plate as the cell culture medium. For example, add 50µl of R1 to a 96-well plate containing 50µl of cell culture medium [Note 3].

6.Shake the plate for 3 minutes, then place it in the dark for an additional 5 minutes to allow lysis.

7.Measure the luminescence signal using a fluorescence plate reader. This reading represents firefly luciferase activity [Note 4].

8.Add an equal volume of R2 working solution to each well of the cell culture plate.

9.Shake the plate for 3 minutes to ensure thorough mixing, then place it in the dark for an additional 10 minutes to allow lysis.

10.Measure the luminescence signal using a luminescence plate reader. This reading represents Nano-Luc luciferase activity.

11.Perform data processing as required by the experiment [Note 5].

1. The luciferase reaction is temperature-sensitive. Reagents and experimental cell plates should be equilibrated to room temperature (22-25°C), and a constant temperature (±1°C) should be maintained during detection. 2. Prepare the R2 working solution fresh as needed. After initial use, aliquot the R1 reagent and R2 substrate for storage at -20°C or lower. Mixing reagents from different batches is not recommended. 3. The volume ratio of cell culture medium, R1 reagent, and R2 reagent should be 1:1:1. Unless validated, altering the detection reagent ratio is not advised. 4. It is recommended to complete plate reading within 1 hour after adding R1 or R2 reagent to obtain optimal results. 5. For data comparison between experimental plates, appropriate control samples should be included as inter-plate internal references. Normalize the experimental data of each plate using the inter-plate internal reference before comparison. Additionally, the reading time after adding R1 and R2 reagents should be consistent across all plates to ensure uniform signal decay rates between plates.