How is luciferase measured?
Luciferase activity is measured using a Luminometer equipped with an automated dual injection system. A volume of 20 μl of cell lysate is transferred to a microcentrifuge tube and placed in the injection chamber.
How is the luciferase protein detected?
In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes.
How big is a luciferase gene?
The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length.
What does the protein luciferase do?
Luciferases are proteins with enzymatic activity that, in the presence of ATP, oxygen, and the appropriate substrate (typically luciferin), catalyze the oxidation of the substrate in a reaction that results in the emission of a photon.
What type of protein is luciferase?
Fluorescent Proteins 101: Luciferase. Luciferases are a class of enzymes capable of catalyzing chemical reactions in living organisms resulting in the emission of photons.
How do you use 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.
How does dual luciferase 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 gene makes luciferase?
The gene for the most common luciferase comes from the firefly, but luciferases from other animals such as the sea pansy Renilla reniformis, the copepod Gaussia princeps, and the ostracod Cypridina noctiluca are also used as reporters.
Is luciferin a protein?
In some cases, the luciferin is a protein known as a photoprotein, and the light-making process requires a charged ion to activate the reaction. The terms luciferin and luciferase both come from a Latin term lucifer, which means “light-bringer.” They are generic terms rather than the names of particular chemicals.
What produces the luciferase protein?
How does a luciferase assay work?
The luciferase assay is useful to study whether a protein of interest regulates a particular gene at the transcription level. When this protein activates transcription, the cell will produce luciferase enzyme. After the addition of a lysis buffer and a substrate, a luminometer quantifies the luciferase activity.
What is dual luciferase assay?