Dual Luciferase Reporter Gene Assay System Protocol

First, we need to understand what luciferase is

Luciferase is the common name for a group of enzymes that catalyze the luminescence of biomolecules using luciferin as a substrate. This enzyme typically combines with oxygen or other cofactors (Mg²⁺, ATP, etc.) in an oxidation reaction that catalyzes the luminescence of luciferin. Finally, visible light is emitted, allowing bioluminescence measurements.

So what is dual luciferase?

Dual luciferase amplifies the signal using two luciferase reporter genes, commonly called firefly luciferase and renilla luciferase.

Firefly luciferase is isolated from the beetle (Photinus pyralis) and has a molecular weight of 61 kDa. Firefly luciferase catalyzes the oxidation of beetle luciferin under the action of ATP, Mg2+, and O2 and emits yellow-green fluorescence at a wavelength of 560 nm (Figure 1).

Figure 1.  Principle of luminescence of firefly luciferase

Renilla luciferase is isolated from Photinus pyralis. Its molecular weight is 36KDa. This enzyme does not require catalysis by ATP or Mg²⁺, only the action of O₂ and coelenterazine is required. It emits blue fluorescence at a wavelength of 465nm.

Figure 2. Luminescence principle of seafood kelsipherase

After understanding it clearly, you should know what the dual luciferase reporter gene detection service mainly does.

Luciferase emits visible light when binding to a substrate to insert a desired gene sequence upstream or downstream of the firefly luciferase gene and construct a firefly luciferase master reporter gene plasmid. At the same time, the umiciitakeluciferase gene must be modified to perform a similar role as the internal reference gene and then co-transfected into cells. After appropriate stimulation or treatment, cells are lysed. After complete reaction by adding an appropriate amount of substrate reactant, luciferase activity is measured and the target gene is determined using the fluorescence intensity. Intensity of action.

So, while you understand the principles, I would like to know where you can apply this service.

Research on promoters

Structural analysis of the promoter, truncation of the promoter, and study of promoter activity by mutation at a specific site. SNP analysis of the promoter.

Research on transcription control

Identify transcription factors and regulatory proteins that regulate gene expression. By introducing mutations and deletions into the promoter sequence, researchers can study their effects on transcriptional regulation.

Post-transcriptional control studies (miRNA)

To determine the target interaction of microRNA, insert the 3'UTR sequence of the mRNA to be tested into a reporter gene vector and co-deliver the microRNA. Decreased luciferase activity marks the target sequence.

Post-translational control (acetylation, phosphorylation, ubiquitination, etc.)

Study of cell signaling pathways

By inserting pathway-specific elements into reporter genes, details of cellular signaling pathways can be monitored.

Protein interaction studies

The principle is that the luciferase protein is divided into a C-terminal part and an N-terminal part, which do not emit light alone, and other proteins are connected. When two proteins interact, they can bind at the C-terminus and N-terminus and emit light.

Small animal live imaging

A living cell in which the luciferase gene is inserted into the chromosome of the cell of interest, an expression line capable of stably expressing the luciferase gene is cultured through cell culture, etc., and inoculated into experimental animals, and the corresponding luciferin is injected from outside the body. A luminescence effect can be created; the more labeled cells, the stronger the fluorescence intensity.

This article mainly describes the experimental principles and applications of dual luciferase reporter genes. So what are our strengths?

We are experienced at the level of experimental manipulation, experimental procedures, and reliable statistical principles to ensure that each set of experiments is reproducible and the experimental data is truly reliable. We have complete supporting testing equipment to further analyze the results, and provide a detailed and complete dual luciferase labeled gene detection experiment report, including experimental data, photos, result analysis, etc.