What is a 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).
How is luciferase assay 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 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 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.
Is a luciferase assay a cell based assay?
The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available.
What is the purpose of a reporter assay?
Reporter gene assays are typically used to measure the regulatory ability of an unknown DNA-sequence. This is done by linking the unknown promoter sequence to an easily detectable reporter gene whose product can be easily detected and quantifiably measured.
What wavelength is luciferase assay?
Therefore, although bioluminescence emitted from deep within an experimental animal can be detected with a sensitive detection system, luciferases that emit light at wavelengths >600 nm have the greatest sensitivity for in vivo imaging applications (2).
How does a reporter gene assay work?
How does luciferase imaging work?
Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling.
What is the purpose of luciferase?
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.
What is the difference between luciferin and luciferase?
Why the name luciferin? The answer to that question and others will take some science as well as some Latin to answer. Luciferase is an enzyme that catalyzes a light-producing biochemical reaction when it is in the presence of oxygen and a naturally occurring substrate called luciferin.
What kind of protein is luciferase?
Dinoflagellate luciferase is a multi-domain eukaryote protein, consisting of an N-terminal domain, and three catalytic domains, each of which preceded by a helical bundle domain.
What is the purpose of the luciferase?
Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly. The scientist produces a construct in which the regulatory region of a target gene is fused with the DNA coding sequence for luciferase (Figure 14.25).
How is luciferase 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.
What do reporter assays identify?
Is luciferase fluorescence or luminescence?
fluorescence. Bioluminescence (left) is emitted from the reaction of luciferase enzyme and its substrate, such as firefly luciferase and luciferin, respectively. Cofactor requirements (e.g., ATP, O2) vary depending on the luciferase used.
How long is luciferase signal?
Current bioluminescence imaging, using commonly available luciferin substrates, only lasts a short time (15–20 min). To avoid repeated administration of luciferase substrate during cancer detection and surgery, a long lasting bioluminescence imaging substrate or system is needed.
What color is luciferase?
Various forms of the enzyme responsible for the fireflies’ luminescence — luciferase — can produce slightly different colours, ranging from red to yellow to green.
How is bioluminescence imaging done?
Bioluminescence imaging (BLI) relies on detection of light from luciferase-expressing cells in a model. This is commonly achieved through implantation of cells engineered to express luciferase constitutively or by use of transgenic animals that express luciferase in one or more tissues of interest.
Why is it called luciferin?
“Luciferin” got its name from the Latin word “lucifer” (meaning “light-bearing”), which is also a source of the word that is sometimes used as a name of the devil.
What type of protein is luciferase?
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 enzyme is luciferase?
Is luciferase magnetic?
This preliminary study shows that luciferase-modified magnetic nanoparticles is a magnetic platform that can be utilized as a possible alternative for QD-based bioimaging, and which also has potential for magnetic cellular manipulation and MRI applications.
Why is luciferase important?
Is luciferase better than fluorescence?
Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later.