Dual Luciferase Reporter Assay Protocol

A luciferase assay is a method used to determine whether or not a protein can activate/repress the expression of a target gene. These assays can identify a functional connection between the presence of a protein and the levels of gene product produced. Dual luciferase assay systems enable the rapid quantification of Firefly and Renilla luciferases in mammalian cell culture samples.

PBS, pipette or multichannel pipette, immunoassay microplates (black is preferred), luminometer detector or full-spectrum microplate reader.

• Mix the Reaction Buffer II (Luciferase) with the lyophilized Luciferase substrate (in the brown dark bottle). Mix thoroughly and keep away from light at -70℃.
  
  
• Mix 5 × Cell Lysis Buffer and ddH₂O in a ratio of 1:4 before use. Keep on ice during experiment.
  
  
• Renilla Substrate is dissolved in ethanol. For initial use, centrifuge briefly and carefully measure the volume of the solution in the tube. If the volume of the liquid is significantly reduced in the tube, add ethanol to bring the total volume back to 200 µl.
  
  
• When using the Renilla Substrate, store on ice. Calculate the actual amount required and mix with the appropriate amount of Stop & Reaction Buffer and Renilla Substrate in a ratio of 50:1. Store at room temperature away from light.
  
  
• The enzymatic reaction is sensitive to temperature. The cell lysis solution and the detection substrate solution should be equilibrated to room temperature before adding sample.
  
  
• Use a luminometer or multi-mode plate reader with luminescence functionality for detection. The value of background signal and sample values may differ between detection instruments. Therefore, it is recommended to optimise the assay and detect the background signal of the substrate prior to experimentation. The values determined on different instruments should not be compared directly. If a full-spectrum microplate reader is used for detection, it is recommended to use an opaque microplate and guarantee a certain interval between the detection wells.

1. Cell Lysis
Discard the cell culture medium and wash the cells twice with PBS. Add the appropriate amount of 1 × Cell Lysis Buffer as recommended in the table below. Incubate or shake for 5 min at room temperature, pipette up and down and transfer the cell lysate to a 1.5 ml centrifuge tube. Centrifuge for 2 min, 12000 × g at room temperature, and collect the supernatant for experimentation.

2. Firefly luciferase activity detection
Add 100 μl of Luciferase Substrate (pre-equilibrated to room temperature) to the detection tube or microplate. Carefully pipette 20 μl of the cell lysate into the test tube or the plate. Mix rapidly and immediately detect the Firefly luciferase reporter gene activity using a luminometer or a full-spectrum microplate reader.

3. Renilla luciferase activity detection
Add 100 μl of freshly prepared Renilla Substrate solution to the above reaction solution. Mix rapidly and immediately detect the Renilla luciferase reporter gene activity.

1. The optimum lysis time may vary for different cell lines. It is recommended to start from 5 min and extended the lysis time to 10 min for complete lysis. After the lysis is completed, do not pipet the cells to prevent the production of air bubbles/foam which may affect enzyme activity.
   
   
2. If the expression level of luciferase is too low, the amount of Cell Lysis Buffer can be appropriately reduced to increase the protein concentration.
   
   
3. In general, the addition of the Stop & Reaction Buffer can inhibit the more than 99% of the activity of Firefly luciferase, however, there may be trace activity left. Therefore, it is recommended to control the RLU value of expression of Renilla luciferase at a level comparable to or slightly higher than that of Firefly luciferase during transfection.
   
   
4. The luminescence intensity is stable for ~ 1 minute after the lysate is in contact with the substrate. When using a single-tube luminometer, the optimal time interval between the mixing of different samples and substrates and the detection on the machine should be as consistent as possible. When using a full-spectrum microplate reader, the cell lysate should be added to the well first, then the detection substrate should be added and tested on the instrument as soon as possible. The measurement time can be set between 1 - 10 sec according to the intensity of the luminescence value. Increasing the detection time will increase the luminescence value of the sample as well as the background at the same time.