TWO-Step Luciferase (Firefly & Renilla) Assay System

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Catalog #
60683
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Description

The TWO-Step Luciferase (Firefly & Renilla) Assay System is designed for high throughput, rapid quantitation of both Firefly and Renilla luciferases in a single mammalian cell culture sample. It comes with enough reagents for 100 assays of 100 μl each in a 96-well plate.

This assay system has several relevant features:

  • Sensitive – highly sensitive detection of Firefly and Renilla luciferase activity.
  • Stable – the luciferase signal output is stable for more than one hour, providing incubation time flexibility.
  • High-throughput – its simple homogeneous protocol minimizes handling steps and supports high-throughput screening applications.
  • Compatibility – works with a variety of common media containing up to 10% serum and phenol red.

Dual Luciferase Assay System

Figure 1: TWO-Step Luciferase (Firefly & Renilla) Assay System protocol overview.
The Firefly Luciferase Reagent is added directly to the cell culture medium. This reagent lyses the cells and contains a substrate that allows Firefly luciferase to generate a luminescence signal. Next, the Renilla Luciferase Reagent is added to the same well. The reagent quenches the Firefly luciferase luminescence and provides the substrate for Renilla luciferase to generate Renilla luciferase luminescence signal. The signal generated by both reactions can be conveniently measured on a luminometer.

Figure 2: Chemical reactions involved in the TWO-Step Luciferase (Firefly & Renilla) Assay System.
A. Chemical reaction involved in signal generation by Firefly Luciferase Reagent Buffer (Component A) and Firefly Luciferase Reagent Substrate (Component B). B. Chemical reaction involved in signal generation by Renilla Luciferase Reagent Buffer (Component C) and Renilla Luciferase Reagent Substrate (Component D).

This product has been cited 41 times.

Synonyms
Luciferin, Firefly luciferase, luciferase buffer, renilla, luciferase detection buffer
Product Info
Storage and Usage
Citations41
Materials Required But Not Supplied
  • Multiwell tissue culture plates compatible with the reading instrument.
  • Mammalian cells express Firefly Luciferase under the experimental promoter and Renilla Luciferase under a control promoter.
  • Appropriate cell culture medium.
  • Laboratory platform shaker.
  • Luminometer
Format

60683-1 (10 ml)

Catalog # Name Amount Storage
  Firefly Luciferase Reagent Buffer (Component A) 10 ml -20°C
  Firefly Luciferase Reagent Substrate (Component B) 100 µl -20°C (Protect from light)
  Renilla Luciferase Reagent Buffer (Component C) 10 ml Room Temp.
  Renilla Luciferase Reagent Substrate 100 µl -20°C (Protect from light)

* Renilla Luciferase Reagent Buffer (Component C) should be thawed at 37°C overnight immediately after receipt. It can be stored at Room Temperature indefinitely

60683-2 (100 ml)

Catalog # Name Amount Storage
  Firefly Luciferase Reagent Buffer (Component A) 100 ml -20°C
  Firefly Luciferase Reagent Substrate (Component B) 1000 µl -20°C (Protect from light)
  Renilla Luciferase Reagent Buffer (Component C) 100 ml Room Temp.
  Renilla Luciferase Reagent Substrate 1000 µl -20°C (Protect from light)

* Renilla Luciferase Reagent Buffer (Component C) should be thawed at 37°C overnight immediately after receipt. It can be stored at Room Temperature indefinitely

60683-3 (1 L)

Catalog # Name Amount Storage
  Firefly Luciferase Reagent Buffer (Component A) 10 x 100 ml -20°C
  Firefly Luciferase Reagent Substrate (Component B) 10 x 1000 µl -20°C (Protect from light)
  Renilla Luciferase Reagent Buffer (Component C) 10 x 100 ml Room Temp.
  Renilla Luciferase Reagent Substrate 10 x 1000 µl -20°C (Protect from light)

* Renilla Luciferase Reagent Buffer (Component C) should be thawed at 37°C overnight immediately after receipt. It can be stored at Room Temperature indefinitely

Background

Firefly luciferase has been used as a sensitive reporter to study a wide range of biological responses. It was first cloned from the North American Photinus pyralis and catalyzes the oxidation of D-luciferin, in the presence of ATP and magnesium, emitting yellow light. This reaction has a high quantum yield and both luciferase and luciferin have low toxicity. These characteristics contributed to Firefly luciferase becoming a commonly used tool. Renilla luciferase, on the other hand, is ATP-independent and converts coelenterazine to coelenteramide in the presence of oxygen, emitting blue light. Both luciferases have a relatively low half-life, versus for instance fluorescent proteins, and allow dynamic studies to be performed. The accuracy of Firefly luciferase reporter in response to a certain stimulus can be improved by the normalization to a control reporter, such as Renilla luciferase reporter, in the same sample.