Learn how the flexstation 3 can be used to accurately and easiy run dual luciferase reporter assays using the integrated fluidics and sensitive optics of the flexstation 3 system. Single, dual, multicolored and realtime luciferase reporter assays. Dualreporter assays enable researchers to obtain additional information from complex systems with minimal effort. Oct 22, 2014 this animation describes the basic concept of reporter assays using luciferase, and explains how a dual luciferase assay works. The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research.
Genecopoeias secretepair system offers the unique advantage of a secreted dualreporter luminescence system, which allows reporter assays in live cells. Dual luciferase reporter system promega 24 well plate. Dual luciferase reporter system promega 24 well plate transfections cells should be transfected with a test reporter firefly luciferase and a normalization reporter driven by a constitutive promoter renilla luciferase prltk thymidine kinase promoter works well at a ratio of 1. In this dual reporter system, both firefly photinus pyralis and renilla reniformis sea pansy luciferase activities are measured in succession within the same well as both are functional enzymes. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Dualluciferase reporter dlr assay system provides an efficient means of performing dual reporter assays. Reporter assay and dualluciferaser promega corporation. Common uses for plasmids expressing luciferase luciferase reporter assay. Application of the dualluciferase reporter assay to the analysis of. To see articles that cite the use of the dualglo luciferase assay system, visit. Secreted dual reporter assay with gaussia luciferase and the. In dualglo luciferase assay protocol, firefly luciferase is measured and quenched, followed by renilla luciferase, in the same well.
The yeast dual luciferase reporter dlr was characterized and shown to be. Transfect cells with pgl34 plasmid and prl vector 50. It uses luciferase enzymes from the firefly photinus pyralis and sea pansy renilla reniformis, measuring their activities sequentially in a single sam. May 26, 2010 this dual luciferase format allowed detecting compound cytotoxicity or offtarget change in expression during drug screening, therefore improved data quality.
This blog is an overview of the luciferase reporter assay focusing on the 4 main types. In the nanodlr assay protocol, firefly luciferase activity is measured first using the oneglo ex luciferase assay reagent. This animation describes the basic concept of reporter assays using luciferase, and explains how a dual luciferase assay works. The nanoglo dual luciferase reporter nanodlr assay system is a homogeneous reagent system that allows you to sequentially detect the activities of firefly photinus pyralis luciferase and nanoluc luciferase nluc from a single sample. Is there anybody willing to share a detailed protocol with me. Protocol for dna transfection and luciferase assay using pfr. This animation describes the basic concept of reporter assays using luciferase, and explains how a dualluciferase assay works. Dual luciferase assay system for rapid assessment of gene. The dual luciferase assay is performed by sequentially measuring the firefly and. I have recently used the dualluciferase reporter assay promega and followed all the specifications of its protocol. The protocol presented here provides details of how to apply the dualluciferase assay to determining the activity of constitutive and inducible. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Improved dualluciferase reporter assays for nuclear receptors.
The dual luciferase reporter dlr assay system provides an efficient means of performing two reporter assays. The cignal reporter assays are available as transfectionready constructs utilizing two reporter systems. The accuracy of firefly luciferase reporter can be improved by normalizing to a control reporter, such as renilla luciferase reporter, in the same sample. Detect dual luciferase expression on the flexstation 3.
Luciferase reporter assay for unlocking ligandmediated. Luciferase reporter assay in drosophila and mammalian tissue. Dual luciferase reporter assay detection on next generation molecular devices author. Each system contains sufficient reagents to perform. Stay notified of promega events, products and news. The dual luciferase reporter dlr assay system provides an efficient means of performing dual reporter assays. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. Dualluciferase reporter assay system technical manual pdf. The luciferase protocol described utilizes the dualglo luciferase assay system from promega which is designed for high throughput assays. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12well dish. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate a stabilized luminescent signal. Luminescent signals generated in the dualluciferase reporter assay system by firefly and renilla. Luciferase reporter assay kit university of michigan. Luciferases are widelyused reporters of biological activity, due to their bright signals and nearly nonexistent background luminescence.
The firefly luciferase reporter is measured first by adding luciferase assay reagent ii. The dualglo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. Grow li cells transformed with pfrwt plasmid dna in appropriate volume of lb or 2xyt media with ampicillin overnight and isolate dna using standard qiagen mini or maxiprep protocol. The firefly luciferase luminescence is measured first by addition of the ffl reagent. 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. The dualluciferase reporter assay or dlr is widely used to study gene transcription and regulation.
The typical luciferase containing plasmid is most often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated regions, or the effect of mutations of these regulatory elements on gene expression. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or. Performing most dualluciferase assays involves adding two reagents to each sample and measuring luminescence following each addition. Bioassay systems dual luciferase reporter gene assay sldl100 allows for the sequential activity measurement of two different luciferases, firefly and renilla, in a single sample.
Since firefly and renilla luciferases use different substrates, both can be measured in the same well using a dual luciferase detection assay. Both gluc and seap are secreted reporter proteins, permitting detection without cell lysis. The dualglo luciferase assay system is a homogeneous reagent system that enables fast and simple quantitation of a stable luminescent signal from two reporter genes in a single sample. Genecopoeias secretepair dual luminescence assay kit is designed to analyze the activities of gaussia luciferase gluc and secreted alkaline phosphatase seap in a dual reporter system. The dual luciferase format is an endpoint assay providing unsurpassed sensitivity, specificity, and signaltonoise ratios. Nanoglo dualluciferase reporter assay system nextgen.
Does not apply to renilla luciferase substrate renlite substrate, whose long term storage is 20 c. You will need phosphatebuffered saline, a luminometer, and a. Dualluciferase reporter assay system promega corporation. On day 3 i perform the luciferase assay using the dual glo kit by promega. The dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum.
Dual luciferase reporter dlr assay certification bmg labtech. Protocol a promotor assay dualluciferase reporter assay. 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. Luciferase assay was performed with dualluciferase reporter assay system kit. Dualluciferase reporter assay system, 1,000 assays. Introduction firefly beetle photinus pyralis luciferase is one of the most popular reporter. In the dlrz assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. The dlr assay is a two step reaction that uses two luciferase enzymes, fire. The dual luciferase reporter assay allows for the sequential measurement of the activity of two different luciferases, firefly ffl and renilla rl, in a single sample.
This requirement has been very conveniently met with the popular dual luciferase assay. The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. Dual reporter assays like this that use either the same or different substrates are commercially available. A second luminescent reporter, renilla luciferase, is often used under the control of a constitutive promoter to normalize for sources of variability such as transfection efficiency and cell number. In the dlr assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. Protocol for dna transfection and luciferase assay using pfrwt plasmid. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Dual spectral 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. Dual luciferase reporter assay kit ba0180 assay genie. Dual luciferase reporter dlr assay system provides an efficient means of performing dual reporter assays. Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Either measuring substrate consumption or overall production, the enzyme assays are used to monitor enzymatic activity.
These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains. Dual luminescence kits and cloning vectors genecopoeia. Some luminometers directly inject the reagents into the cell lysate. Thus, dual reporter assays often allow more reliable interpretation of the experimental data by reducing extraneous influences. The dualglo luciferase assay system is designed to allow highthroughput analysis of mammalian cells containing genes for firefly and renilla luciferases, grown in 96 or 384well plates. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dual spectral analyses. The dual luciferase reporter dlr assay systemac provides an efficient means of performing dual reporter assays. Reporter assays use bioluminescent reporters whose detection is based on the enzymatic reaction with an enzyme, luciferase, with the substrate luciferin. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. Single and dualreporter assays cells are inherently complex, and the data available from a single reporter may be insufficient for achieving reliable results. In the dlr reporter assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. Luciferase reactions were performed according to manufacturers instructions using titration curves of a purified.