How do luciferase reporter assays work?

August 28, 2019 Off By idswater

How do luciferase reporter assays work?

A Luciferase Reporter Assay. 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. If your protein activates the expression of the target gene, the amount of signal produced increases.

How long does luciferase last?

Luciferase enzymes with flash kinetics have maximum sensitivity because of high signal intensity, although the emitted light also rapidly decays. Conversely, enzymes with glow kinetics are less sensitive but stably emit light for at least 60 minutes.

Where is luciferase found?

insect fireflies
Luciferase is a light-producing enzyme naturally found in insect fireflies and in luminous marine and terrestrial microorganisms.

What color is luciferase?

The color of light that is emitted by luciferase is highly dependent on the amino acids that surround the luciferin. PDB entry 2d1s (on the left) is the structure of luciferase from a Japanese firefly. It normally emits a greenish-yellow light.

What causes luciferase?

Luciferases are enzymes that produce light when they oxidize their substrate. 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.

Does GFP affect luciferase?

In our hands, there were no differences in luciferase data using either GFP-tagged or un-tagged vectors to overexpress our protein of interest.

Who named luciferin?

The Discovery of Luciferin and Luciferase by Raphaël Dubois Dubois used bioluminescent clams and cold water to make a glowing paste. He split the paste into two parts. When he heated the first sample to near boiling, the glow immediately stopped.

How was luciferin named?

“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’s the best way to use the luciferase assay?

1. Add 1 mg coelenterazine to 1 ml of acidified methanol (add 50 µl of concentrated HCl to 10 ml methanol). 2. Aliquot and store at -80°C for up to 4 weeks. 3. For use, thaw tube and add 25.41 µl of coelenterazine stock solution to 40 ml DPBS (1.5µM final concentration).

How is luciferase produced in a cultured cell?

This construct, as well as a construct coding for the protein, is introduced into cultured cells. If the protein is able to activate transcription, the cell will produce the luciferase reporter. The amount of luciferase produced can be quantified using a luminometer.

Which is the luciferase reporter 1000 assay system?

E4030) is not suitable for these applications. The Luciferase Reporter 1000 Assay System (Cat.# E4550) was designed to meet the needs of users who perform a large number of assays, particularly in 96-well plates. The system contains sufficient reagents to perform 1,000 luciferase assays (100µl per assay).

How is luciferase quantified in a luminometer?

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. If your protein activates the expression of the target gene, the amount of signal produced increases.