luciferase assay

Intro

Genetic reporting assay is an experimental technique that is widely used for cell response or gene expression research due to external stimulation in prokaryotic or eukaryotic cells.

In particular, the dual-reporter assay uses two independent reporter systems to increase the accuracy of the experiment.
One of them is an experimental reporter, whose response degree varies depending on the experimental conditions.
The other is internal control, which does not change according to the experimental conditions, and is used to normalize the value for the experimental reporter.

Normalized data can be used by correcting differences that may occur during the experimental process, such as transfection efficiency (gene insertion efficiency), cell viability, cell lysis, and pipetting.
Luciferase Assay System uses firefly luciferase and Renilla luciferase, which are separated from two types of fireflies, as experimental and control reporters, respectively.

Firefly luciferase uses luciferin as a substrate to convert it into Oxyluciferin through oxidation and emits 560 nm of light. Renilla luciferase uses coelenterazine as a substrate to catalyze the oxidation process and emit 480 nm of light. As such, the two enzymes show different reactions using different substrates, so one enzyme.
It can be used simultaneously in the system. (Figure 1)

Figure 1. Bioluminescent Reactions Catalyzed by Firefly and Renilla Luciferase

The experimental method 
Luciferase Assay System
1. After adding a substrate for firefly luciferase and reacting it, measure activity (luminescence) for firefly luciferase
2. Add Stop & Glo reagent, react
3. Measurement of activity (luminescence) against Renilla luciferase
4. Normalization: Calculate Firefly: Renilla ratio for each well
In this application note, Synergy HT Multi-Detection Microplate Reader is used
Introducing the activity measurement method of Dual-Glo luciferase.


Result and Discussion
Figure 2 shows the experimental results measured by varying concentrations to measure each protein activity.
For both proteins, the luminescence value also increases as the concentration increases.

 

Figure 2. Firefly Luciferase and Renilla Luciferase Concentrations Kabits

This is the result of maintaining the concentration of Renilla luciferase and stepwise diluting only the concentration of Firefly luciferase to confirm the reaction in which the activities of Firefly and Renilla luciferase occur independently.
Regardless of the concentration of Firefly, it can be seen that the luminous value of Renilla Luciferase remains constant.
(Figure 3).
Since the activity of the two proteins does not affect the other, firefly is used to see the results according to the experimental conditions, and Renilla can be used as internal control.

Figure 3. Firefly and Renilla Singals from Common wells

Figure 4 shows the results of Figure 3 as a ratio.
The X-axis is the result of the Molar ratio (Firefly/Renilla) of Firefly luciferase and Renilla luciferase.
The Y axis is the result of the signal ratio (Firefly/Renilla) of Firefly luciferase and Renilla luciferase.
If you don't have a firefly in the same well, and you have a lot of Renilla, it's a very low molar ratio.
Conversely, the molar ratio of a high value is calculated when the amount of firefly is large and the amount of Renilla is small, and the molar ratio can be expressed in proportion to the signal ratio as follows.

Figure 4. Comparison of the Firefly/Renilla Signal Ration to Firefly/Renilla Enzyme Molar Ratio

Firefly and Renilla light emission signals are stable and long-lasting, so the dual-glo assay is
You can measure different gene reporter samples using techniques.