How do you perform luciferase assay?

To perform a luciferase reporter assay, you will need a DNA plasmid to express the protein that you hypothesize could affect transcription. You will also need to use another DNA plasmid with the regulatory element (or a target promoter region) fused with the DNA coding sequence for a luciferase enzyme.

What does a luciferase assay tell you?

A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene.

How does dual luciferase reporter assay work?

The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc).

What is FLUC and Rluc?

The relative Fluc/Rluc ratio following transfection of various constructs into MAC-T cells. (A) Schematic diagram of modified pGL3-control vector with firefly luciferase (FLuc) coding region that was transiently transfected into MAC-T cells. Renilla luciferase expression construct (RLuc) was cotransfected as control.

What is a promoter assay?

Promoter analysis can be done by analyzing the expression levels of RNA. The assumption is that genes that have similar expression levels have similar transcriptional regulation control and common binding sites. This knowledge is used we have to find promoter regions on genome sequences.

What are luciferase used for?

Luciferases are widely used in biotechnology, for bioluminescence imaging microscopy and as reporter genes, for many of the same applications as fluorescent proteins.

Which is a substrate for luciferase?

Luciferases comprise a group of enzymes that emit light in the presence of oxygen and a substrate (luciferin).

How do you measure promoter activity?

Methods to study promoter activity commonly are based in the expression of a reporter gene from the promoter of the gene of interest. Mutations and deletions are made in a promoter region, and their changes on couple expression of the reporter gene are measured.

Why is luciferase used in medicine?

Luciferase enzymes from a variety of different organisms have been used for BLI studies of viral infection and other biologic processes (Table 1). Firefly (Photinus pyralis) luciferase, optimized for expression in mammalian cells, is the most commonly used luciferase for in vivo imaging.

What can luciferase be used for?

The power of luciferase has been harnessed by scientists to devise reactions whose light output is used to monitor biological processes including gene expression, biomolecular binding, and cell viability.

Who discovered luciferase assay?

While DuBois discovered the reaction between luciferin and luciferase in 1885, it was not until the late 1940s when the luciferase protein was first extracted and purified firefly lanterns by Drs. Green and McElroy. Using this process, they isolated the enzyme and determined its conformational structure.