Is luciferase reporter assay?
A luciferase reporter assay is a test that investigates whether a protein can activate or repress the expression of a target gene using luciferase as a reporter protein (Carter & Shieh, 2015). This bioluminescence directly corresponds with the effect of the protein on expression of the target gene.
What is the most common use of luciferase reporter gene?
Luciferase reporter assays are widely used to investigate cellular signaling pathways and as high-throughput screening tools for drug discovery (Brasier et al. 1992, Zhuang et al. 2006).
What is the purpose of luciferase assay?
A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene.
What is a luciferase reporter gene?
A commonly used reporter gene is the luciferase gene from the firefly Photinus pyralis. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg(++), yielding a fluorescent product that can be quantified by measuring the released light.
What is the role of a reporter gene?
Reporter genes are genes that enable the detection or measurement of gene expression. They can be fused to regulatory sequences or genes of interest to report expression location or levels.
How does a reporter assay work?
A bioluminescent reporter assay consists of both a luciferase reporter enzyme and a detection reagent that provides the enzyme substrate. When the reporter enzyme and detection reagent are combined, the light emitted is proportional to reporter gene expression levels and is detected using a luminometer.
Is GFP a reporter gene?
Since the cloning and enhancement of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria (4, 7, 9, 27–29, 41, 46), GFP has been widely used as a reporter gene.
How does a reporter gene work?
Reporter genes include genes that code for fluorescent protein and enzymes that convert invisible substrates to luminescent or coloured products.
What is an example of a reporter gene?
In eukaryotes, gene fusions use different reporter genes. For example, yeast reporter genes include CUP1, a gene that enables yeast to grow on copper-containing media, URA3, a gene that kills yeast when growing on 5-fluorouracil, and ADE1 and ADE2, two genes that synthesize adenine.
What makes a good reporter gene?
The ideal reporter gene should be absent from the cells used in the study or easily distinguishable from the native form of the gene, assayed conveniently, and have a broad linear detection range.
How is the luciferase reporter assay used in cell biology?
This reporter assay can be used to study gene expression as well as other cellular components and events that are involved in gene regulation.
How are luciferase assays different from Chip and EMSA?
Unlike the ChIP or EMSA assays, which only assess the ability of a protein to interact with a region of DNA, a luciferase assay is able to establish a functional connection between the presence of the protein and the amount of gene product that is produced.
How do you perform a reporter assay on a gene?
To perform the reporter assay, you clone the regulatory region of your gene-of-interest (X) upstream of the luciferase gene in one of these expression vectors, introduce that resulting vector DNA into cells, and let the cells grow for a period of time.
How is luciferase measured in a dual Glo assay?
In Dual-Glo® Luciferase Assay protocol, firefly luciferase is measured and quenched, followed by Renilla luciferase, in the same well. Performing most dual-luciferase assays involves adding two reagents to each sample and measuring luminescence following each addition.