The name was first used by raphael dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. Reporter protein assays use colorimetric, radioactive or luminescent methods to measure enzyme activity present in a cell lysate. Assays based on luminescence are very popular for several reasons. The dualluciferase reporter dlr assay system contains two different luciferase reporter enzymes that are expressed simultaneously in each cell. In addition, we present a detailed protocol for the in vivo detection of extracellular atp in mice using luciferase transfected reporter cells. The protocol is written to accommodate a 96well plate, but this can just as easily be.
For example, renilla luciferase is not secreted, and thus the pierce renillafirefly luciferase dual reporter assay kit will not work for this type of timecourse assay. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. The 293ft producer cells were grown in white walled, flat surface 96well plates and were cotransfected with ar expression vector and are reporter vectors according to the manufacturers instruction invitrogen, cat. The dual reporter luciferase method is well suited for studying gpcr modulators in a highthroughput format. The system, provided with cell culture lysis reagent cclr, also can be used for reporter quantitation. Viafect transfection reagent transfection assay e4981. Reagents for luciferase reporter assays thermo scientific turbofect transfection reagent deliver dna plasmids and expression vectors into eukaryotic cells with this highly efficient, easytouse, nonimmunogenic transfection reagent. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses.
To determine if a protein is able to activate or suppress transcription of a gene of interest, a scientist uses recombinant dna technology to produce a construct in which the genes promoter is placed adjacent to a luciferase reporter gene. Nanoglo dualluciferase reporter assay nanodlr system. The bioglo luciferase assay system provides a highly sensitive, robust, homogeneous reagent for the detection of firefly luciferase reporter gene expression in the adcc reporter bioassay. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a. The firefly luciferase reporter assay provides rapid results of cellular transfections 14. Application noteluciferase assay and bio tek synergy microplate readers. A protocol for reporter quantitation in mammalian cells.
Highthroughput screening hts assays used in drug discovery frequently use reporter enzymes such as firefly luciferase fluc as indicators of target activity. Reporter labeled cell lines are useful for monitoring various biological functions. A bioluminescence reporter assay for retinoic acid control of. Spectramax duoluc reporter assay kit molecular devices. What is the reason for the effect on renilla signal in the. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate a stabilized luminescent signal.
Indigos aryl hydrocarbon receptor ahr assay is a cellbased genetic reporter assay that utilizes the luciferase reporter gene aryl hydrocarbon receptor agonist and antagonist assays functionally linked to an ahrresponsive promoter. Luciferase reporter assay system for deciphering gpcr. Mar 10, 2017 luciferase reporter gene assays are a type of reporter gene assay that utilizes the light generated by one of a family of enzymes known as luciferases. A commonly used reporter gene is the luciferase gene from the firefly. Then, rip was performed to assess the combination between mir1443p and linc00461 or kpna2, and dual luciferase reporter assay was used to validate the targeted relationship between mir1443p and kpna2. Luciferase assays have since become a gold standard in gene expression analysis and a luciferase gene one of many available to choose from is now a common feature in reporter plasmids. General considerations the nanoglo dualluciferase reporter assay system is designed to be used with many media types and has been validated for use with the following culture media containing 010% serum. Typically, a promoter region containing 2 to 3 kb of a 5. Because of the requirement of atp, firefly luciferases have been used extensively in biotechnology. Detection of e2finduced transcriptional activity using a dual luciferase reporter assay chapter pdf available in methods in molecular biology clifton, n. Improved assay performance by using destabilized luciferase reporter. The dual luciferase fireflyrenilla assay system is designed to be used for highthroughput, rapid quantitation of both firefly and renilla luciferases from a single sample in mammalian cell culture.
Furthermore, transwell was employed for the examination of cell invasion and migration in breast cancer. A specific mechanism for nonspecific activation in. A bioluminescence reporter assay for retinoic acid control. Engineered luciferase reporter from a deep sea shrimp. Aid 411 qhts assay for inhibitors of firefly luciferase. Due to the longlasting signals, the luciferase assay, on the other hand, provides the opportunity to take multiple measurements over a longer time period. It involves classical luciferase protocol in which the chimeric plasmid luciferase gene and the target promoter is transfected into the cells. Transfection basics, principle, mechanism and optimization of mammalian cell. Reporter constructs rely on the insertion of a genetic regulatory. Luciferase is particularly useful as a reporter in living cells and organisms. Reporter genes molecular biology, stem white board animat. Luciferase cell culture lysis reagent promega, madison, wi, sonicated and vortexed, followed by centrifugation for 10 minutes at 4c and 15,000 rpm. Quantitation of luminescent signal from each of the luciferase reporter enzymes may be performed immediately following lysate preparation without the need for dividing samples or performing additional treatments.
Dualluciferase reporter assay system harvard university. Development of reporter assays to measure regulation of gene expression will facilitate understanding of regulatory mechanisms involved in this process. The are reporter hep g2 cell line is designed to monitor nrf2 antioxidant response pathway. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of hts data. This bioassay record aid 411 belongs to the assay project probe summary for inhibitors and stabilizers of firefly luciferase, which is associated with the summary aid 2309 and a total of 18 additional bioassay records in pubchem. The cell contains a firefly luciferase gene under the control of are antioxidant response element stably integrated into hep g2 cells. Mechanism of ptc124 activity in cellbased luciferase assays of nonsense codon suppression douglas s. A wide variety of luciferase enzymes have been discovered, although the general mechanism of the lightproducing reaction for all of the enzymes is the oxidative decarboxylation of the luciferase substrate in the presence of o 2 to yield photon emission light. Luciferase reporter assays were performed by cotransfecting hek293 cells with 8.
What is the reason for the effect on renilla signal in the luciferase assay and what is the mechanism of transcription without promoter. In rare cases, this activity appears counterintuitive. The luciferase assay kit is intended for the detection of the expression of firefly luciferase and is not suitable for the assay of luciferase from other origins. All you wanted to know about luciferase but were afraid to ask using luciferase experimentally. In vitro transcribed rnabased luciferase reporter assay. The improved reporter assays showed larger assay dynamics in shorter assay time than using regular luciferase. Firefly luciferase, one of the most commonly used reporters, produces light by catalyzing a bioluminescence reaction that oxidizes dluciferin in the presence of oxygen and atp. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of. Firefly luciferase is an exquisitely sensitive reporter with a halflife of. Mechanism of ptc124 activity in cellbased luciferase assays of nonsense codon suppression. The polya tail length was analyzed by rnase h digestion and northern blotting as previously described. A further advantage of using geneencoding rna is that, together with the luciferase reporter gene ivt rna, any other antigen or vaccine ivt rna of interest can be cotransfected. In this perspective, we provide evidence for stabilization of luciferase. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately.
The glow is widely used as an assay for luc expression, which acts as a reporter for the activity of any genetic regulation that controls the expression of its gene. We show that although ptc124 and related compounds cause apparent activation in cellbased nonsense codon suppression assays using a fluc reporter, they are inactive in the same assay when a different bioluminescent reporter, renilla reniformis luciferase rluc is used as the reporter. Dec 10, 2018 bioluminescent reporter assay for monitoring er stress in human beta cells. This animation describes the basic concept of reporter assays using luciferase, and explains how a dualluciferase assay works. Kinetic analysis and modeling of firefly luciferase as a. Choose your luciferase reporter gene firefly luciferase or renilla. Allard and kevin kopish, promega corporation luciferase reporter technology gives the researcher unparalleled sensitivity, dynamic range, versatility and ease of use when investigating questions that involve gene regulation. Using a model system of exogenous or endogenous receptors in hek293 cells, we demonstrated that all four major g protein subfamilies and downstream pathways can be studied in one luciferase reporter assay format. Previously, we have shown that the luciferase reporter vectors pgl4 series containing minimal promoter and destabilized luciferase gene improve the responsiveness of camp response element to g s coupled receptor 8 fan f, wood kv. The luciferase reporter assay kit is suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter. Luciferase assay was performed with dualluciferase reporter assay system kit. Dualreporter assay using two secreted luciferase genes.
This assay was introduced into shsy5y cells and used to demonstrate the role of rar. The sensitivity was adjusted as necessary to ensure light levels remained within the instruments detection limits. The luciferase from the deep sea shrimp, oplophorus, suggested a route for achieving these capabilities. An androgen response element driven reporter assay for the. The firefly luciferase reagent is first added to the cells in medium directly. Luciferase mrna transfection of antigen presenting cells. For the conventional luciferase reporter assay, each indicated luciferase reporter and expression construct was cotransfected together with 400 ng of a renilla luciferase reporter construct prlbasic, as an internal control for transfection efficiency, into nih3t3 cells using lipofectamine plus reagent invitrogen. Pdf detection of e2finduced transcriptional activity using. These cells either constitutively or inducibly express a reporter gene such as gfp, rfp, luciferase, or lacz. We exploited this stressinduced splicing mechanism to generate a lentivirus vector containing a stressinducible gaussia luciferase reporter plvcmvxbpglucbcpuro fig. Dual reporter assay luciferase is widely used as a reporter due to its sensitivity and linear range. The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect proteinprotein interactions in vitro, in cellulo, and in vivo.
To assay the samples, 10 al of the cell lysate were added to 100 al of the luciferase assay buffer, swirled briefly, and placed in the luminometer. It has broad applications across various fields of cell and molecular biology wherever you want to measure or track expression of a cloned gene. The enzyme catalyses the oxidation of firefly luciferin, requiring oxygen and atp. Luciferase reporter mice for in vivo monitoring and ex vivo. The dlr and dualglo assays provide rapid and convenient means for achieving greater control over the biological significance of reporter data by differentiating genetic responses of. In vitro luciferase activity was quantified using the luciferase assay system promega, madison, wi. Luciferasebased genetic reporter assays provide sensitive methods for. Rna interference rnai is a mechanism that inhibits gene expression before a gene.
Identification of a novel circadian clock modulator. Luciferase assay system protocol promega corporation. Cellbased assays validation and comparison of singlestep. Signosis has developed p53 luciferase reporter hela stable cell line, in which luciferase activity is specifically associated with the activity of p53. Transient transfection and luciferase assay 20121212. Reporter genes are essential tools in the study of gene expression and regulation, as they are designed to function as surrogates for genes involved in key cellular processes and disease. This chapter describes the luciferase assays that are available to monitor yaptaz activity in cell lines and to study their regulation, including the choice for the normalizer, a description of the main yaptazresponsive luciferase reporters used so far by the community, and technical notes and experimental considerations on the most appropriate positive controls. Reporterlabeled cell lines are useful for monitoring various biological functions. The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. Dualspectral luciferase reporter assays using luciferase enzymes with nonoverlapping luminescence spectra can be measured simultaneously by filterbased detection in a single sample using a single reagent. Use of luciferase probes to measure atp in living cells.
Both words are derived from the latin word lucifer meaning lightbringer. Using luciferase reporter assays to screen for gpcr modulators. Nov 08, 2019 all you wanted to know about luciferase but were afraid to ask using luciferase experimentally. The dualluciferase reporter dlr assay and dualglo assay enable the sequential measurement of both firefly and renillaluciferases from one sample. Bioglo assay reagent contains a new luciferase substrate, resulting in a reagent that is more stable and more tolerant to sample components than. Luciferase reporter gene assay dualspectral luciferase dual reporter luminescence renilla gaussia firefly introduction reporter assays have long been used to investigate regulatory mechanisms of gene expression in response to environmental stimuli in a variety of organisms. Maguire, and james inglese2 nih chemical genomics center, national institutes of health, bethesda, md 208923370. The dual luciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Luciferase reporter assay kit university of michigan. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible.
When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. M mir26a mimic or negative control mimic with 500 ng of psicheck2 reporter plasmids using lipofectamine 3000 thermo fisher scientific and performing the dual luciferase reporter assay system promega after 48 hours. Preparation of are reporter response luciferase assay. Performing the nanoglo dual luciferase reporter assay 3. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. A bioluminescence reporter assay was developed that expresses firefly luciferase under the control of the glur1 5. Therefore, the cell line can be used as a reporter system for monitoring the activation of p53 triggered by stimuli treatment, enforced gene.
Reporter gene assays are an important tool for monitoring cellular events associated with gene expression, gene regulation, and signal transduction. Luciferase reporter assays allow you to investigate promoter activity by measuring light output from luciferase enzyme that is expressed under the control of your promoter of interest. Dec 12, 2012 transient transfection and luciferase assay 20121212. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. Luciferase reporter mice for in vivo monitoring and ex. The importance of bioluminescence in enabling a broad range of highthroughput screening hts assay formats is evidenced by widespread use in industry and academia. Arebinding protein zfp36l1 interacts with cnot1 to. Mathematical model of the firefly luciferase complementation.
Aryl hydrocarbon receptor assay ahr indigo biosciences. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Transfection guide overview of transfection methods. It has broad applications across various fields of cell and molecular biology wherever you want to measure or. Bioluminescent reporter assay for monitoring er stress in. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay.
Over 600,000 random and chemically diverse small molecule compounds were screened. The pheromoneresponsive luciferase reporter assay for rgs4 inhibitors was conducted as a singledose assay at a final compound concentration of approximately. However, cypridina and gaussia luciferases are secreted. The luciferase reporter assay was performed by nanoglo luciferase assay systems promega. Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The firefly luciferase and the renilla sea pansy luciferase can discriminate between their respective bioluminescence substrates and do not crossactivate. Atcc is continually accessioning and creating new reporter cell lines to quicken the pace of your research. Learn how luciferase can be used in concert with fluorecent proteins in nano lanterns. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. Jan 27, 2015 the luciferase reporter gene assay, based on the responsibleelementregulated expression of luciferase, has been widely applied in the highthroughput functional assay for many targets because of. Mar 03, 2009 firefly luciferase is an exquisitely sensitive reporter with a halflife of. Mechanism of ptc124 activity in cellbased luciferase assays.
Mechanism of ptc124 activity in cellbased luciferase. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. Dual reporter assays like this that use either the same or. Here we describe a protocol to study mrna translation using an in vitro transcribed rnabased luciferase reporter assay in poxvirusinfected cells.
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