ONE-Step™ Luciferase Assay System
Luciferase is the general term given to a class of oxidative enzymes which catalyze reactions that give off light, a process known as bioluminescence (Figure 1). Enzymatic activity is directly proportional to the expression of luciferase in the cells and can be quantified upon addition of the appropriate substrate. In the luciferase reaction, the cells are lysed with luciferase substrate containing D-luciferin. Firefly luciferase expressed in reporter cells catalyzes the conversion of D-luciferin to oxyluciferin using ATP as a co-substrate, and in the presence of Mg2+, in a reaction that gives off light. The amount of light is proportional to the amount of luciferase present in the reaction. Luminescence is read using a luminometer.
Description:
The One-Step™ Luciferase Assay System is designed for high-throughput, sensitive quantitation of firefly luciferase activity in mammalian cell culture. The reagent consists of two components, a Luciferase Reagent Buffer (Component A) and Luciferase Reagent Substrate (Component B). Component A and Component B are combined to form a working solution that contains all the necessary reagents for cell lysis and luciferase quantitation.
- Sensitive - highly sensitive detection of firefly luciferase activity.
- Stable - the signal is stable for more than two hours, providing flexibility regarding incubation time.
- Convenient - simple one-step, homogeneous protocol.
- High throughput - the one-step protocol minimizes handling steps to support high-throughput screening applications.
- Compatibility - works well with a variety of common media containing 0-10% serum and phenol red.
- Instrumentation - does not require a luminometer with injectors.
More about the supplier BPS Bioscience
TWO-Step Luciferase (Firefly & Renilla) Assay System
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.
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.
More about the supplier BPS Bioscience
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).
INDIGlo Luciferase Detection Reagent Kit
The INDIGlo Luciferase Detection Reagent (LDR) is formulated to deliver a steady, long-lived luminescence signal from mammalian reporter cells expressing firefly luciferase. For convenience, the reagent is provided ‘ready to use’, without the requirement to first re-constitute powder-form components.
How is INDIGlo used?
INDIGlo LDR works equally well in quantifying expressed luciferase activity from 96- or 384-well format reporter assays. The resulting light emission is stable over a 2 hr period (Figure 1), thereby making INDIGlo LDR an ideal, economical detection reagent for both low- and high-throughput batch processing of assay plates.
INDIGlo LDR is formulated as a 2x-concentrated reagent, thereby allowing greater versatility in its use. The 2x-concentrated reagent will be used when using 384-well or 1536-well assay plates that are processed using a homogenous assay strategy.
Alternatively, when processing 96-well assay plates either a homogenous or non-homogenous assay strategy may be used. For convenience, this kit provides an optimized Dilution Buffer for use in diluting INDIGlo LDR when performing non-homogenous assays.
Components
INDIGlo Luciferase Detection Reagent kits provide two reagents in equal volumes:
- INDIGlo LDR, formulated as a 2x-concentrated reagent
- Dilution Buffer, for optional use if performing non-homogenous assays
Performance
INDIGlo LDR performance from both Homogenous and Non-Homogenous assays. Dose-response analyses of Human AhR (INDIGO #IB06001, 96-well assay) was performed using the reference agonist MeBIO. AhR reporter cells were plated across two white assay plates, MeBIO treatment media were added, and the assay plates incubated for 24 hr. Plates were then processed with luciferase detection reagents in either a.) a homogenous or b.) a non-homogenous assay format. Resulting assay metrics are also shown from using another extended-glo detection reagent (Vendor X).
INDIGlo LDR potency is stable following repeated freeze and thaw cycles. INDIGlo LDR was frozen at -20°C and thawed either 1, 2 or 3 times, then used to quantify luciferase activity from HEK-293:luc cells cultured in a white 96-well assay plate. No loss of reagent potency is observed between these conditions.
Manuals
| Filename | Download |
|---|---|
| TM_INDIGlo-Luciferase-Detection-Reagent-v1.2.pdf |  |