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- Bioluminescent Reporter Assays_Overview
Bioluminescent Reporter Assays_Overview
Bioluminescent Reporter Assays_Overview
Luciferase reporter assays are essential tools in cell biology for analyzing gene expression, biosensors, and protein interactions. They offer high sensitivity and a broad dynamic range, making them superior to fluorescent assays. These assays are widely used in promoter studies, gene regulation, and high-throughput screenings (HTS).
Promega provides three main types of luciferases: Firefly, Renilla, and NanoLuc®. NanoLuc® is particularly noted for its high signal intensity and small size, making it ideal for live-cell protein interaction studies.
NanoLuc® is a genetically optimized luciferase from deep-sea shrimp, offering 150 times brighter luminescence than other luciferases. It is stable, with a half-life over two hours, and retains activity at high temperatures, making it suitable for sensitive and robust assays.
The design involves cloning reporter vectors, transfecting cells, and detecting signals. Promega provides various reporter vectors for transcriptional and post-translational studies.
Luciferase reporters are used in promoter studies, signal transduction analysis, compound screening, RNA interference, and protein interaction assays. NanoLuc® enhances these applications with its high sensitivity and brightness.
These assays analyze promoter activity and genetic elements by correlating luciferase expression with transcriptional activity. Dual-reporter assays improve data reliability and normalization.
Promega's luciferase technologies, particularly NanoLuc®, provide innovative solutions for cellular biology research, offering high sensitivity and versatility for various applications.
This section discusses promoter deletion analysis to identify regulatory regions within a promoter sequence. Truncated variants of a promoter are tested using a dual-reporter assay, with normalization against a control reporter to adjust for experimental variability.
The document describes pGL4 and pNL1 plasmids as optimized promoterless reporter vectors for regulatory analysis in mammalian cells. These vectors have cryptic regulatory sequences removed to avoid co-regulatory effects, and the codon usage of luciferase genes is adapted for high expression efficiency in humans.
A vector map of a co-reporter plasmid is provided, which uses the thymidine kinase (TK) promoter for constitutive and moderate expression levels of the control luciferase reporter in mammalian cells.
Dual-reporter assays are recommended for transient promoter studies to increase data consistency. Combinations such as Firefly/Renilla and NanoLuc®/Firefly are suggested, with NanoLuc®-based assays noted for their sensitivity at physiologically relevant expression levels.
Reporter gene assays are highlighted for their ability to quantify signaling pathway activities under various conditions. These assays involve cloning response element repeats into reporter vectors upstream of a minimal promoter.
The document details the use of Rapid Response™ Reporters, which improve signal-to-background ratios by preventing intracellular reporter protein accumulation.
Rapid Response™ reporters are shown to increase signal-to-background ratios, providing faster and more pronounced responses to stimuli compared to unmodified reporters.
G-protein coupled receptors (GPCRs) are involved in various signaling pathways. Upon stimulation, different types of GPCRs activate distinct pathways, leading to the activation of transcription factors like CREB, which bind to response elements to initiate gene transcription.
GPCR signal transduction can be studied using response element reporter vectors. These vectors help in screening GPCR modulators and ranking agonists and antagonists by measuring luciferase activity.
Cells respond to stress factors by activating specific pathways to minimize damage. Promega offers RE reporter vectors to study these responses.
Cytokines regulate cell proliferation and differentiation. Reporter vectors are available to study cytokine-mediated signal transduction.
Coincidence luciferase reporters are used in HTS to identify modulators of cell signaling pathways.
RNA interference is a natural mechanism in eukaryotic cells that regulates gene expression post-transcriptionally.
siRNA and miRNA are processed by the enzyme Dicer, forming a complex with proteins like Argonaut to create the RNA-induced silencing complex (RISC).
Luciferase reporters are used to measure RNAi activity. They help identify and optimize siRNA sequences, detect miRNA/siRNA activity, and investigate miRNA target sequences and transcript stability.
- Identification and optimization of siRNA sequences for gene knock-downs.
- Detection and quantification of miRNA/siRNA activity.
- Investigation of transcriptional control and miRNA target sequences.
The pmirNanoGlo vector is used for measuring miRNA activity, while the psiCHECK™-2 vector is designed for siRNA optimization.
NanoLuc® luciferase-based sensors measure protein stabilization in response to signaling pathways.
1. NanoBRET™ Technology
Used for monitoring PPIs in live cells, kinetic measurements, screenings, and biosensor generation.
For sensitive detection of PPIs in living cells, allowing real-time or endpoint assays.
1. NanoBRET™ Target Engagement (TE)
Used for determining drug candidate binding characteristics, selectivity screens, and target validation.
Reporter assays for protein stabilization, degradation, secretion, and receptor internalization.
The document provides detailed information on the generation and detection of HiBiT-tagged proteins using the NanoBiT® HiBiT system, as well as the application of GloSensor™ technology for real-time measurement of intracellular activities.
- Used for monitoring protein interactions, quantifying protein expression, and studying receptor internalization.
- Utilizes a genetically altered luciferase for intracellular protein sensing, enabling real-time measurement of cAMP, cGMP, and protease activity.
- FuGENE® and ViaFect™: Non-liposomal reagents for efficient transfection of various cell types without compromising cell viability.
The document provides comprehensive insights into advanced tools for protein tagging, detection, and transfection, highlighting their applications and benefits in molecular biology research.
The document provides a detailed comparison and analysis of various transfection reagents and luciferase assay systems.
ViaFect™ Transfection Reagent is designed for transfection of adherent, suspension, primary, and stem cells.
Promega's luciferase detection assays are optimized for use with mammalian cells, offering both flash-type and glow-type assays.
Nano-Glo® Luciferase Assay System is highlighted for its high sensitivity and stability.
The document provides a comprehensive guide to selecting appropriate transfection reagents and luciferase assays based on specific research needs.
The document provides detailed descriptions and comparisons of various luciferase assay systems used for quantifying luciferase activity in mammalian cells.
This is a homogeneous, one-step assay designed for high sensitivity and compatibility with high-throughput applications.
ONE-Glo™ offers improved signal intensity and stability compared to Bright-Glo™.
This system features a novel chemistry that extends signal stability to 2 hours.
Steady-Glo® provides an extremely long signal stability of over 5 hours.
This flash-type assay is highly sensitive and provides high signal intensity and linearity over a wide concentration range.
Renilla-Glo™ follows an add-and-read principle, providing a stable luminescent signal with a half-life greater than 1 hour.
The document also discusses dual luciferase assays, which measure the activity of two different luciferase reporters in a single sample.
The document provides a detailed comparison and description of various luciferase assays, including Firefly, Renilla, and NanoLuc® luciferases.
- Luciferases Firefly + Renilla: Non-homogeneous format with 5 steps, high sensitivity, and a medium Z’ factor.
- Firefly + NanoLuc®: Homogeneous format with 2 add-only steps, high sensitivity, and a high Z’ factor.
- Nano-Glo® Dual-Luciferase® Reporter Assay System: Designed for detecting firefly and NanoLuc® luciferase activities in a single sample.
- Dual-Luciferase® Reporter Assay System: Allows sequential detection of firefly and Renilla luciferase activities.
- Dual-Glo® Luciferase Assay System: A homogeneous add-only assay for firefly and Renilla luciferase activities.
- Nano-Glo® Live Cell Assay System: Enables dynamic real-time measurement of NanoLuc® or NanoBiT® activity in living cells.
- NanoBRET™ Nano-Glo® Substrate: Used for detecting NanoLuc® activity in NanoBRET™ assays.
- High sensitivity and signal stability make these assays suitable for various research applications.
- Compatibility with different well formats enhances their utility in HTS.
The document provides detailed information on various luciferin substrates and detection systems used for measuring luciferase activity in living cells.
- VivoGlo™ Luciferin: A potassium salt of D-luciferin used for detecting firefly luciferase activity.
- Luciferin-EF™: An endotoxin-free sodium salt D-luciferin suitable for living organisms.
- ViviRen™ Live Cell Substrate: Used for detecting Renilla luciferase activity.
- EnduRen™ Live Cell Substrate: Provides stable luminescence for up to 24 hours.
- GloMax® Luminometers: Designed for high sensitivity and broad detection range.
- GloMax® Discover and Explorer Systems: Offer modular designs with capabilities for luminescence, fluorescence, and absorbance measurements.
- Detailed specifications for each product, including catalog numbers, are provided for easy reference.
This document provides a comprehensive overview of various cloning vectors, reporter vectors, and assay systems used in molecular biology, particularly focusing on NanoLuc® and related technologies.
The document lists several cloning vectors designed for creating custom NanoLuc® fusion vectors.
Various reporter vectors are detailed, including those for oxidative stress, cAMP/PKA signaling, calcium signaling, and more.
The document outlines multiple assay systems, including single and dual reporter gene assays.
A selection of transfection reagents is provided, including TransFast™ and FuGENE® HD.
The document describes NanoBRET™ assays for protein-protein interaction studies and NanoBiT™ assays for live-cell applications.
Live-cell substrates for real-time measurements are included, such as Nano-Glo® Live Cell Assay System.
The document also lists various substrates and reagents for specific applications, including caspase activity detection and cAMP/cGMP measurement.
The document provides a list of Nano-Glo® HiBiT Detection Systems and GloMax® Detection Instruments.
The Nano-Glo® HiBiT systems are available in different volumes and configurations.
Promega products are intended for specific uses as indicated on product labels.
The document lists contact details for Promega's corporate headquarters, manufacturing facilities, branch offices, and distributors worldwide.
For more information on Promega products and worldwide contacts, users are directed to visit the Promega website.
Catalog excerpts
Reporter Assays for versatile applications Gene Regulation | Cell Signaling Pathways | HTS Screens | RNA Interference Protein Stability | Protein:Protein Interaction | Receptor Interaction Biosensors | Bioassays | Bioluminescent Imaging
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The Brightest Luciferase Reporter Smartphone image of a 384-well plate with NanoLuc®-expressing cells.
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Supporting science around the world With a portfolio of more than 3,000 products covering the fields of genomics, protein analysis/expression, cellular analysis, drug discovery, and genetic identity, Promega is a global leader in providing innovative solutions and technical support to scientists in academic, industrial and governmental settings. Promega provides products globally through its 15 branches and over 50 distributors. Serving more than 100 countries, Promega supports laboratories with its molecular tools, technical support, and customer service. Promega was first certified to ISO standards...
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Table of Contents 1 Introduction Bioluminescent Reporter Assays 6 1.2 NanoLuc® - Renilla - Firefly : A Comparion of Luciferases 7 1.3 NanoLuc® - The Brightest Luciferase Reporter 8 1.4 Experimental Design of Reporter Assays 9 2.1 Promoter Studies with Dual-Luciferase Reporter Assays 12 2.2 Signal Transduction - Analysis of Signaling Pathways using Luciferase Reporters 14 2.3 Compound HTS - Coincidence Luciferase Reporters 22 2.5 Protein Stability Sensors Based on NanoLuc® Luciferase 28 2.6 Protein:Protein Interaction (PPI) Assays - NanoBRET™/NanoBiT®...
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6 Dual Assays for the Quantification of Two Luciferases in a Single Sample 56 6.1 Dual Assays Combining NanoLuc® and Firefly Luciferase 58 6.2 Dual Assays Combining Firefly and Renilla Luciferase 60 7 Live-Cell Substrates for Dynamic Real-Time Measurements & Imaging 62 7.1 Live-Cell Substrates for the Detection of NanoLuc® Luciferase 63 7.2 Live-Cell Substrates for the Detection of Firefly Luciferase 65 7.3 Live-Cell Substrates for the Detection of Renillla Luciferase 66 8 Detection with GloMax® Luminometers 68 9.4 NanoBRET™ Protein:Protein Interaction...
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Luciferase Reporter Assays 6 NanoLuc®- Renilla - Firefly: A Comparision of Luciferases 7 NanoLuc® - The Brightest Luciferase Reporter 8 Experimental Design of Reporter Assays 9 1.1 Luciferase Reporter Assays Luciferases are widely used as reporter genes across various fields of cell biology, including gene expression at the transcriptional, translational, and posttranslational level as well as the generation of biosensors and protein interactions in live cells. The term luciferase refers to a class of enzymes catalyzing a light-emmitting reaction by oxidation of their respective substrate....
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1.2 NanoLuc® – Renilla – Firefly: A Comparison of Luciferases Luciferases are well suited for setting up highly sensitive and easy-to-quantify reporter assays. Traditionally, a combination of firefly and Renilla luciferase is applied to study gene regulation. The firefly luciferase (Fluc) originates from the American beetle Photinus pyralis, while Renilla luciferase (Rluc) derives from the sea pansy Renilla reniformis. By contrast, the newly developed and highly optimized NanoLuc® luciferase (Nluc) originates from the deep-sea shrimp Oplophorus gracilirostris. Its markedly increased signal intensity...
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1.3 NanoLuc® – The Brightest Luciferase Reporter NanoLuc® luciferase (Nluc) is a genetically optimized luciferase that has been developed by directed enzyme evolution of a luciferase from the deep-sea shrimp Oplophorus gracilirostris. Nluc catalyzes an ATP-independent conversion of its optimized substrate, furimazine, yielding luminescence 150-times brighter than FLuc or RLuc (Fig. 1.2). Furthermore, the Nluc substrate furimazine displays enhanced stability with a half-life greater than two hours and a lower background activity, which both open up a new field for bioluminescence imaging. With...
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1.4 Experimental Design of Reporter Assays The experimental design is critical to the success of any reporter assay. Generally, it can be divided into the following key steps: 1 cloning of reporter vector constructs, 2 transfection & cultivation of cells, and 3 signal development & detection (Fig. 1.3). Usually, the most time consuming step is the generation of a reporter construct whose respective design depends on the particular question to be addressed (Fig. 1.4). Studies on transcriptional regulation – e.g. promoter studies – require the insertion of a regulatory DNA sequence into a promoterless...
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2.1 Promoter Studies with Dual-Luciferase Reporter Assays 12 2.2 Signal Transduction - Analysis of Signaling Pathways using Luciferase Reporters 14 Cellular Stress Response 19 2.3 Compound HTS - Coincidence Luciferase Reporters 22 Measure miRNA Activity with pmir Reporter Vectors 26 Measure siRNA Activity with psiCHECK™-2 Reporter Vector 27 2.5 Protein Stability Sensors Based on NanoLuc® Luciferase 28 NanoBRET™ Target Engagement 34 2.8 Protein Tagging and Detection System - NanoBiT® HiBiT 36 2.9 Live-Cell Biosensors for cAMP, cGMP or Protease Activity - GloSensor™...
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Applications of Luciferase Reporters Introduction – Applications of Luciferase Reporters Luciferase-based detection systems are broadly applied by scientist from academia as well as pharmaceutical industries due to its unparalleled sensitivity, dynamic range, versatility and ease of use. Starting from the 1990s, Promega has been continuously developing luciferase-based products and technologies. The development of the highly effective NanoLuc® Luciferase (Nluc) opened up novel exciting possibilities and improvements to luminescence applications, including monitoring of protein stability and detection...
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Applications of Luciferase Reporters 2.1 Promoter Studies with Dual-Luciferase Reporter Assays Application Studies on promoter activity, promoter deletion analysis, or analysis of single nucleotide polymorphisms (SNPs) within promoter sequences. Promoter deletion analysis Promoter region Description One common application of luciferase reporter genes is analyz- Regulating region ing the function of cis-acting genetic elements such as promot- ers (“promoter bashing”). Typically, deletions are made within a promoter region, and their effects on coupled expression of a luciferase reporter gene are...
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