Firefly Luciferase

1. The Brief Introduction of Firefly Luciferase What is Firefly Luciferase? Luciferase reporter system is a kind of reporter system which USES luciferin as substrate to detect firefly luciferase activity. The bioluminescence emitted by luciferin during oxidation can then be measured using a luminometer, also known as a luminometer or liquid flash tester. Luciferin and luciferase, a bioluminescent system, can detect gene expression very sensitively and efficiently. It is a method to detect the interaction between transcription factors and DNA in the promoter region of the target gene. DNA transcription into RNA is a key process of gene expression, and gene expression regulation mainly occurs at the transcription level. One of the trends in the field of gene molecular biology is to gradually shift from the analysis of gene structure and function to the cis-acting elements and transcription factors and their transcriptional regulation mechanism. Dual-luciferase reporter assay (DR) has become an effective method to investigate the involvement of transcription factors in gene regulation. Through the analysis of promoter DNA fragments, the trans-activation ability of promoter binding elements was verified, and the molecular mechanism of transcription factors in signal transduction was explored. Transcription factor, also known as trans-acting factor, is a protein molecule with a special structure that regulates gene expression. Some transcription factors bind only

2. to specific sequences in their target promoters. These specific sequences are known as cis-acting elements. The DNA binding domain of transcription factors and cis-acting elements covalently bind, thus inhibiting or enhancing gene expression. Luciferase reporter assay (luciferase assay) is an important method to detect the specific sequence of these transcription factors and their target promoters. The Principle of Firefly Luciferase Its principle is summarized as follows: (1) construct a reporter plasmid, such as pgl3-basic, that inserts a specific segment of the target promoter into the front of the luciferase expression sequence. (2) transcription factor-expressing plasmids to be detected were co-transfected with reporter plasmids in 293 cells or other related cell lines. If the transcription factor can activate the target promoter, the luciferase gene will be expressed, and the expression amount of luciferase is proportional to the action intensity of the transcription factor. (3) specific luciferase substrates were added and luciferase reacted with the substrates to produce fluorescence. The activity of luciferase could be determined by detecting the intensity of fluorescence, so as to determine whether the transcription factor could interact with the target promoter fragment. The Development of Firefly Luciferase Dual luciferase reporter testing: advanced co-reporter testing techniques combining firefly and Marine coelenteric luciferase in quantifying gene expression with firefly luciferase usually use a second reporter to reduce experimental variation. However, traditional co-reporter genes (such as CAT, meg-gal, and GUS) are not convenient enough, because their respective test chemistry and processing require different detection characteristics. Promega offers an advanced dual-reporter technology that combines firefly luciferase testing with Marine coelenteric luciferase testing. Double luciferase reporter gene test system, combined with pRL vector system, expresses the second reporter gene luciferase in Marine coelenterate, and performs double luciferase reporter gene test in a single tube, which is rapid, sensitive and simple. The system also provides PLB lysates for cleavage of mammalian cells cultured in porous plates, without the need to operate a single sample. For researchers using firefly luciferase reporter gene vectors. The double luciferase reporter testing system will allow them to experience the convenience of the system.