What is a dual reporter assay?
The Dual-Luciferase® Reporter (DLR™) Assay System provides an efficient means of performing two reporter assays. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated simultaneously by adding Stop & Glo® Reagent to the same sample.
How do you perform luciferase assay?
Luciferase Reporter Assay To perform the luciferase assay, 100 µ l of each plate lysate is added to 360 µ l of luciferase assay buffer (Table I) in a luminometer vial. The reactions are started by the injection of 200 µ l of the 0.2 mM luciferin solution (Table I), and light output is measured for 20 sec at 25°C.
What is 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.
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 is the luciferase enzyme?
Luciferases are enzymes that use a substrate called luciferin, along with oxygen and ATP, in an energetic process that produces light—like the yellow glow of fireflies.
How many cells are in luciferase assay?
The assay yields a linear correlation between cell number and luminescence: up to 50,000 cells per well for 96-well plates within a measurement window of zero to 15 minutes.
Are reporters assay?
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.
What is a reporter molecule?
Reporter genes are those genes that when introduced into target cells (e.g., brain tissues, cancer, and circulating white cells) produce a protein receptor or enzyme that binds, transports, or traps a subsequently injected imaging probe.
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). This bioluminescence directly corresponds with the effect of the protein on expression of the target gene.
How long do luciferase assays take?
This is a flash-type assay that requires luminescence to be measured immediately after adding the detection reagents to the luciferase sample. Firefly signal decays over the course of about 12 minutes, while Renilla signal decays over the course of about 2 minutes, although this may vary depending on enzyme levels.