UA-Glo® Dual Luciferase Assay System

Dual luciferase reporter genes have wide applications in the study of mammalian cell functions, including cell metabolism, signaling pathways, transcription factors, gene functions, drug screening, etc. In the dual luciferase reporter system, one luciferase serves as the experimental reporter gene to indicate the target under study; the other luciferase acts as the control reporter gene to normalize the activity of the experimental reporter gene, thereby minimizing experimental errors caused by transfection efficiency, cell number, cell lysis effect, sample volume, etc.

The UA-Glo Dual Luciferase Reporter Gene Assay Kit has the advantages of high sensitivity, good stability, good repeatability, and convenient use, and its overall performance surpasses similar foreign products. In the same sample, first add R1 reagent to detect the activity of firefly luciferase, then immediately add R2 reagent to quench the firefly luciferase while detecting the activity of Renilla luciferase. The entire process for detecting one sample is completed within tens of seconds. If a multi-well plate is used for detection, the required experimental time is even shorter. During data analysis, the experimental results are normalized by calculating the ratio of the relative fluorescence intensity of the experimental luciferase to that of the control luciferase.

Reagent R1 is filled in 10 ml or 100 ml brown bottles. R2 includes R2 buffer and R2 substrate. R2 buffer is filled in 10 ml or 100 ml brown bottles, and R2 substrate is dispensed into 2ml spiral tubes. The specific specifications are as follows:

1. Plate the cells to be tested at an appropriate density in a 96-well or 384-well cell culture plate, and transfect with appropriate plasmids or stable cell lines carrying relevant dual-luciferase expression vectors. It is recommended to use white plates.
2. Perform relevant treatments on the cells according to project requirements and continue culturing for an appropriate period of time.
3. Take out the detection buffer and substrate, and equilibrate at room temperature for 20 minutes.
4. Take out the cell culture plate to be tested and equilibrate at room temperature for 20 minutes.
5. Prepare detection reagents. R1 reagent can be used directly. R2 substrate is a 100x concentrated solution; before use, dilute it with R2 buffer to make R2 1x working solution. Note to avoid light.
6. Add 50 µl of R1 reagent to 100 µl of cells in a 96-well plate, or 10 µl of reagent to 20 µl of cells in a 384-well plate, gently shake for 2 minutes, and place in the dark to continue lysis for 5-8 minutes.
7. Read the fluorescence signal on a luminescence microplate reader. This is the firefly luciferase activity reading. Save the data.
8. Add 50 µl of R2 1x working solution to the above reaction plate wells and gently shake for half a minute.
9. Read the fluorescence signal on a luminescence microplate reader. This is the Renilla luciferase activity reading. Save the data.
10. Perform data processing and analysis.
11. It is recommended to store unused reagents at -20°C.

1. Temperature: Unless otherwise specified, luciferase detection is performed at room temperature (22-25°C). Temperature has a significant impact on enzyme reactions.
2. Reagent dosage: It is not recommended to arbitrarily change the dosage of reaction reagents without strict verification.
3. Plate reading time: Please perform plate reading according to the time recommended in the instruction manual. It is advisable to complete the plate reading within 10-30 minutes to obtain the best results.
4. Inter-plate internal reference: If inter-plate data comparison is required, it is recommended to set up two wells of positive controls without added compounds in all reaction plates as inter-plate internal references. The readings of each plate should first be corrected (normalization) using the inter-plate internal reference readings, and the corrected data should then be subjected to downstream processing and analysis.
5. Reaction plates: The level of fluorescence reading may cause significant interference with the readings of adjacent wells, mainly due to the spillover of fluorescent signals. If necessary, opaque-bottom white plates or black plates can be used for experiments or verification.
6. Reagent mixing: It is not recommended to mix reagents from different batches, reagents that have been activated but not used up and stored, and reagents with significantly different storage conditions.
7. Aliquot storage: Dispense and store reaction reagents according to the instruction manual to ensure the stability of the reagents.