The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. In the dlr assay, the activities of firefly photinus pyralis and renilla renilla reniformis or sea pansy luciferases are measured sequentially from a single sample. Problem with luciferase reporter assay researchgate. The firefly luciferase reporter is measured first by adding. Assay lysates for luciferase activity and protein concentration. Storage reconstituted luciferase assay reagent should be stored in aliquots at 20c for 1 month of 70c for up to 1 year. The luciferase assay system yields linear results over at least eight orders of magnitude.
Oct 27, 2008 the dual luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. The dualglo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. Dualluciferase reporter assay system 10pack e1960 from. Bioluminescent reagent system for rapid quantitation of firelfy and ranilla luciferase reporter gene expression in transfected cells key features high sensitivity and wide detection range. The oneglo luciferase assay system employs new assay chemistry to provide a robust, homogeneous assay. Dual luciferase reporter assay system instructions for use of products e1910 and e1960. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains. Addition of oneglo ex reagent lyses cells and provides the substrates to measure firefly luminescence.
Dualluciferase reporter assay system protocol promega. Open access improved dualluciferase reporter assays for nuclear receptors aileen paguio, pete stecha, keith v. What is a luciferase assay and what is it useful for. Dual luciferase assay data analysis issues protocol online. The composition of a noncommercial dual luciferase enzyme assay system was exactly as described previously. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Cell pellet of cells with luciferase and no luciferase as a control. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. Comparative analysis of dualluciferase assay technologies. Compatible with routine laboratory and hts formats.
The firefly luciferase reporter is measured first following the. Our method is adapted from a protocol generously provided by ron johnson at the nih and a previously described noncommercial dualluciferase assay 20. The dualluciferase reporter assay system was used exactly according to the manufacturers protocol. Prior to starting use recombinant luciferase to create a standard curve for light output of your luminometer. Goldbios illumination duraluc lyophilized firefly hts assay kit contains a proprietary formulation that modifies and stabilizes the enzymatic reaction to produce a long las. Systems contain one vial each of brightglo luciferase assay substrate and buffer. 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. Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level.
This report describes the optimization and evaluation of a simple singlestep lysis protocol to measure luciferase bioluminescence from genetically modified plasmodium falciparum. However, the assay is designed to allow for higher throughput processes given a considerably more stable luminescent signal for both reporters and homogenous assay format with no washing or preconditioning required. Use biorad assay for protein concentrations in 20ul of. The dual luciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Protocol for dna transfection and luciferase assay using. In a pinch, all three of the dual kits can be used to measure firefly luminescence, but the second reporter must match the kit. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. I performed a dual luciferase assay using pgl3 firefly luc as experimental reporter and prltk renilla luc as internal control promega assay kit. A protocol for combined photinus and renilla luciferase. Dual luciferase reporter assay and dual luciferase reporter assay system quick protocol fb024 pdf 73 kb english.
This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. Prepare the luciferase reagents in the interim, following promega instructions. Promega luciferase assay systems fisher scientific. Store at 20c, where it is stable for up to 6 months. It has broad applications across various fields of cell and molecular biology wherever you want to measure or. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. After following the protocol for the dual glo luciferase assay system and assaying luminescence, i normalized the data to empty vector control as recommended by promega. Luciferase dual reporter assay kit bioassay systems 877. The thermo scientific pierce luciferase dual assays deliver high sensitivity in a simple onestep detection protocol. The dualluciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Highthroughput functional screening using a homemade dual. Dual luciferase gene reporter assays to study mirna function. Just looking at the data, it seems that there is a significant difference in my experimental promoter fragment compared to empty vector controls.
In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a. The nanoglo dual luciferase reporter nanodlr assay system allows sensitive detection of firefly and nanoluc luciferases. If not assaying directly in the culture plate, transfer lysate to an opaque white plate 50 l for 96 well plates, 12. Apr 05, 2019 because of this, a fireflyrluc system should be measured using the dualglo luciferase assay system e2920 or dualluciferase reporter assay system e1910. It uses luciferase enzymes from the firefly photinus pyralis and sea pansy renilla reniformis, measuring their activities sequentially in a single sample. Dualluciferase reporter assay system harvard university. Allow the luciferase substrate and cell lysate containing luciferase to equilibrate to room temperature before use.
The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. Protocol for dna transfection and luciferase assay using pfr. Calculation of transfection efficiency for dual luciferase. Our method is adapted from a protocol generously provided by ron johnson at the nih and a previously described noncommercial dual luciferase assay 20. Here, we present a rapid and sensitive assay based on the classical dual luciferase reporter technique which can be. A luciferase assay takes advantage of the innate bioluminescent properties some organisms exhibit, most notably the firefly. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. Wood and frank fan promega corporation, madison, wisconsin, usa abstract. Nanoglo dualluciferase reporter assay protocol promega. Dual luciferase reporter dlr assay system provides an efficient means of performing dual reporter assays. This assay yields a glow reaction with a halflife of approximately 1 hr figure 7. The firefly assay buffer lyses cells and provides atp and luciferin, the substrates of firefly luciferase. We established a quantitative reporter gene protocol, the prluc assay system, allowing the sequential measurement of photinus and renilla luciferase activities from the same extract.
Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. Potent inhibition of firefly luciferase coupled with the highintensity luminescence of nanoluc luciferase maximizes sensitivity for detection of both. Systems include stop and glo buffer and substrate, one vial of luciferase assay substrate, 10 ml luciferase assay buffer ii, and 30 ml passive lysis 5x buffer. A new luciferase substrate has enabled promega to create a novel reagent that is more robust and stable when reconstituted, and less aromatic than standard reagents created with luciferin. It uses luciferase enzymes from the firefly photinus pyralis and sea pansy renilla reniformis, measuring their activities sequentially in a single sam. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube.
Here, we present a rapid and sensitive assay based on the classical dualluciferase reporter technique which. Dual luciferase reporter assay chemistry continued in the dlr assay chemistry, the kinetics of the renilla luciferase reaction provide a stabilized luminescent signal that. Do not wash 293t cells since they detach easily place 200. Just looking at the data, it seems that there is a significant difference in my experimental promoter fragment compared to. Homogeneous also compatible with prelysis sample process. Nuclear receptors play important roles in many cellular functions through control of gene transcription. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Both words are derived from the latin word lucifer meaning lightbringer. Dual luciferase gene reporter assays to study mirna.
Instructions for use of products e2920, e2940, e2980. The most common scientific assays utilizing luciferase are reporter assays in which transcriptional activation. The superlight dualluciferase reporter gene assay allows for the sequential measurement of the activity of two different luciferases, firefly ffl and renilla rl, in a single sample. In particular, promega has developed a firefly luciferase reporter system to study the regulation of gene expression in mammalian cell culture. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Less than 10 20 moles of luciferase have been detected under optimal conditions. Allow cells to incubate in treatment media for 30 min at 37c 5 % co2. The firefly can convert luciferin to oxyluciferin in the presence of the enzyme luciferase to emit light. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. 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.
Application of the dualluciferase reporter assay to the. Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors. Luciferase assay substrate in 10 ml of luciferase assay buffer. 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. Open access improved dualluciferase reporter assays for. The dual glo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. 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. Prepare treatment medias, thaw cms depending on the assay 2. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately.
The nanoglo dualluciferase reporter nanodlr assay system allows sensitive detection of firefly and nanoluc luciferases. After following the protocol for the dualglo luciferase assay system and assaying luminescence, i normalized the data to empty vector control as recommended by promega. Dualluciferase reporter assay system measures firefly and renilla luciferases from a single sample. Protocol for dna transfection and luciferase assay using pfrwt plasmid. The dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Improved dualluciferase reporter assays for nuclear receptors. Calculation of transfection efficiency for dual luciferase assay. Alternatively, the dual luciferase assay can be performed directly in the culture plate if cells were cultured in opaque plates. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a single sample. It is also a large target class for drug discovery. 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 luciferase assay is one of the most wellknown and convenient reporter assays. The dual glo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. Open access improved dual luciferase reporter assays for nuclear receptors aileen paguio, pete stecha, keith v. The firefly luciferase luminescence is measured first by addition of the ffl reagent. The dual luciferase reporter dlr assay system provides an efficient means of performing dual reporter assays. This dualluciferase format allowed detecting compound cytotoxicity or offtarget change in expression during drug screening, therefore improved data quality. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the prluc assay system was formulated under the aspect of full compatibility with standard methods for protein. Some luminometers directly inject the reagents into the cell lysate. 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. Luciferase reporter gene detection kit luc1 technical. Simultaneous dualemission detection of luciferase reporter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism. Weve made big improvements to our luciferase assay kits, with simplified assay setup, improved working solution stability, and high sensitivity and linearity. May 26, 2010 this dual luciferase format allowed detecting compound cytotoxicity or offtarget change in expression during drug screening, therefore improved data quality.
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