A sensitive reporter assay with longlived luminescence for batch processing in. The triple luciferase assay reagents and protocol described here permit analysis of all three luciferases in the same sample of cell lysate and. Pdf multiplexing fluorogenic esterasebased viability assay. A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene. Luciferase assays and biotek synergy microplate reader. Use biorad assay for protein concentrations in 20ul of. Sep 01, 2006 such systems have found widespread application through the availability of commercial assay kits the dual luciferase reporter assay system, cat. Highly sensitive dual luciferase detection with the. Matt carter, jennifer shieh, in guide to research techniques in neuroscience second edition, 2015. It is based on the murine plasmodium berghei species transformed with luciferase. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 phone 6082744330 fax 6082772516. Illuminating insights into firefly luciferase and other.
Two proteins of interest, a bait and prey, which are genetically fused to amino and carboxyterminal fragments of renilla luciferase, are tr. Assay lysates for luciferase activity and protein concentration. But when coenzyme a has been initially added to the assay buffer. Adjust volumes accordingly for other plate formats. Luciferase assay reagent can be purchased ready to use cat. Do not store luciferase assay reagent with dry ice. Dual luciferase reporter gene assay kit ka3784 products. Includes technical manuals, technical bulletins, product information sheets, protocol cards and automated protocols for highthroughput systems. Dispense 100l of the luciferase assay reagent into luminometer tubes, one tube per sample.
Some luminometers directly inject the reagents into the cell lysate. Dualluciferase reporter assay system technical manual. Allow the luciferase substrate assay buffer mixture to reach room temperature. Reporter genesmolecular biology, stemwhite board animation. Detect dual luciferase expression on the flexstation 3. Bioglo assay reagent is functionally tested for performance in the adcc reporter bioassay and is intended for use with this or other bioassays. Protocols can also be modified for manual injection using the same or related system. The sensitivity of firefly luciferase assay can vary depending on several factors described in assay conditions below. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Pdf gene assay with thermo jorma lampinen academia. Read the entire protocol before performing the assay. Secreted dual reporter assay with gaussia luciferase and the red. Substrate and firefly luciferase assay buffer is stable for 2 months at.
This kit is most commonly used with cell supernatants, but can also be used with cell lysates. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. Lumino firefly luciferase assay is designed for rapid and high sensitive detection or quantification of firefly luciferase reporter enzyme activity from cultured bacterial or mammalian cells. Luciferase assay system perelman school of medicine at.
Readytoglow secreted luciferase reporter assay protocol. Fast and easy, accurate and linear cell assay kit for the. The dual luciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. The following protocol was optimized using adherentsuspension cells grown in a 96well tissue culture plate. Luciferase assay, alternative extractions freeze and.
Bioglo assay reagent contains a new luciferase substrate, resulting in a reagent that is more stable and more tolerant to sample components than standard luciferase assay reagents. Prepare the luciferase substrate working solution by mixing the contents of 1 bottle of substrate a with 20 ml of substrate b. A split luciferase complementation assay to study proteinprotein interactions within arabidopsis protoplasts in 96well plates is described in this protocol. To see articles that cite the use of the dualglo luciferase assay system, visit. Dual luciferase reporter dlr assay certification bmg labtech. To assess the effect of ca on the transcriptional activity of foxm1, luciferase assay was used as previously described 26. Luciferase assay system quick protocol fb026pdf 102 kb english. Pdf simultaneous examination of cellular pathways using. Determination of luciferase activity in arabidopsis. Product information sheet live cell luciferase assay kit. The dlr assay is a two step reaction that uses two luciferase enzymes, fire. This is a detailed protocol for the in vivo zebrafish luciferase assay as previously described.
Readytoglow secreted luciferase reporter assay protocol ataglance pt39022 this protocol is provided for use with the readytoglow secreted luciferase reporter assay cat. E1910, promega, madison, wi, usa and alternative protocols based on the same principles. When reconstituted, it has the capacity to lyse cells, reduce the autoluminescence of the coelenterazine substrate, and produce a stable signal halflife greater than 60 minutes at 22c. Reagents can be used with injectionbased protocols or with cell lysates made with passive lysis buffer. A the dual luciferase assay protocol is illustrated. Dual luciferase reporter assay system technical manual. Luciferase reporter gene assay, high sensitivity sigmaaldrich. This dual assay includes sequential measurement of these two luciferases.
Do not wash 293t cells since they detach easily place 200. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. An improved singlestep lysis protocol to measure luciferase. Luminescent signals generated in the dual luciferase reporter assay system by firefly and renilla. For manual luminometers, the substrate solution is mixed by hand with the. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a. Without this normalization procedure, our data would have falsely. Our secreted dual reporter assay should be an attractive alternative to the. Introduction firefly beetle photinus pyralis luciferase is one of the most popular reporter.
Abstract an in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with agrobacterium tumefaciens is described. If the same lysate is to be used both for luciferase readings and for sdspage gel and western blotting, then it is best to add protease inhibitors to the 1x passive lysis buffer provided with the luciferase kit. Two proteins of interest, a bait and prey, which are genetically fused to amino and carboxyterminal fragments of renilla luciferase, are transiently expressed in protoplasts. Luciferase assay protocol reading luciferase and renilla signal using promega dual luciferase reporter assay note. Therefore their activities can be measured with high sensitivity in the same samples figure 1 without the usual quenching step needed to differentiate. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or. All you wanted to know about luciferase but were afraid to askusing luciferase experimentally. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. A preconfigured protocol in softmax pro software was used with the parameters shown in table 1. Transient dualluciferase assay combined with a glucocorticoid.
Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. Functional assay description the leeporter luciferase assay reagent was specifically formulated to use with our leeporter luciferase reporter cell lines expressing an optimized intracellular renilla luciferase, which was designed to produce highly sensitive signal and. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. Dualluciferase reporter assay system protocol promega. When stored as directed, the kit is stable for 1 year.
Dual luciferase reporter dlr assay certification bmg. The optimal luciferase assay conditions will need to be measure d in each experiment to maximize the luminescence signals. 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. Detection of luciferase reporter assays bmg labtech. Immediately, divide the luciferase substrate working solution into aliquots and store at 70 c. Firefly luciferase is a widely used reporter to study gene regulation and function. Different applications of the assay have been described in two publications weger et al. Learn about our tools that are helping researchers develop diagnostics and vaccines for the sarscov2 virus. Consecutive detection of firefly and renilla luciferase activities in a single sample. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Leeporter luciferase assay reagent test application. 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.
The luciferase assay system is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. For kits e1500, e1501, e4030, e4530 and e4550, prepare luciferase assay reagent lar by adding luciferase assay buffer 10ml for e152a and 100ml for e152b to the vial of lyophilized luciferase assay substrate. The thermo scientific pierce luciferase dualspectral assays provide a simple onestep detection protocol based on the use of pairs of luciferase enzymes that are spectrally resolved. Luciferase assay systems instructions for use of products e1483, e1500, e1501, e1531, e4030, e4530 and e4550. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. 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. In vivo zebrafish luciferase assays a practical guide 1. 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. A protocol for combined photinus and renilla luciferase. Here, we describe a protocol that includes two alternate data collection methods to quantify luminescence results based on agrobacterium. Linear results are seen over at least eight orders of magnitude of enzyme concentration, and less than 10 20 moles of luciferase can be measured under optimal conditions. The renillaglo luciferase assay system is a singleaddition reagent that generates a glowtype signal with renilla luciferase.
The system contains sufficient reagents to perform 1,000 luciferase assays 100l per assay. Luciferase reporter assay kit university of michigan. Growth conditions, cell numberswell and other factors may affect results. It is a very sensitive reporter due to the lack of any endogenous luciferase activity in mammalian cells or tissue. Nanoglo dualluciferase reporter assay system nextgen. Visit, to download or request copies of the following mate rials. Reagent, that combine to suppress coelenterazine autoluminescence. The dual luciferase reporter assay or dlr is widely used to study gene transcription and regulation. Development of a highthroughput gaussia luciferase reporter. Jun 28, 2010 a split luciferase complementation assay to study proteinprotein interactions within arabidopsis protoplasts in 96well plates is described in this protocol. Stemssheaths of 14dold seedlings were cut into approximately 0. Note the luciferase substrate assay buffer mixture is best if used within one month when stored at 20c or within one year when stored at 70c. The biolux gaussia luciferase assay kit contains the reagents necessary for assaying gaussia luciferase gluc activity. Robust firefly luciferase assay with improved performance for high and ultrahighthroughput applications.
L 10 vials luciferase assay substrate lyophilized l 10. The firefly luciferase activity is measured first by adding luciferase assay reagent ii lar ii. We routinely use the dual luciferase kit from promega, but alter the protocol for lysis depending on the goals of the assay. In addition, a stable reporter protein allows detection of genome damage. Thaw luciferase assay reagent at temperatures below 25c and mix well before use. Here we share a multiplex assay protocol that allows sequential measurement of cell viability cell number and luciferase activity of the same sample in. The split luciferase complementation assay springerlink. Oz biosciences luciferase assay kit instruction manual. The assay protocol was run in wellmode from the gen5 data analysis software biotek instruments, inc. 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. Pdf multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters.
For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube. A protocol for measuring the activity of firefly photinus pyralis and. Hepatic human transformed cells hepg2, pretreated for 18 hours with the compound to investigate, are infected with freshly dissected p. With a preconfigured protocol to simplify data acquisition and analysis, softmax pro software completes this total solution for fast, sensitive, and reliable dual luciferase reporter assay results. The spectramax duoluc reporter assay has been performance optimized on the spectramax i3x reader with injector cartridge. Protoplasts were prepared according to the protocol of bart et al. Jun 15, 2020 protocols are provided on how to prepare dna components and destination vector plasmids, design synthetic dna, perform assembly cloning of new transcriptional reporter elements, implement multipartite synthetic assembly cloning of single. For a general introduction to the theory of the readytoglow secreted luciferase reporter system, please refer to. Addmixmeasure assay for nanoluc luciferase with 2 hour halflife in most applications. Dualluciferase reporter assay system harvard university. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate. In this protocol we describe a modified expression assay using transgenic plants expressing the luciferase gene under the. A novel triple luciferase reporter assay permits measurement of. Gaussia luciferase is a reporter luciferase from the marine copepod gaussia princeps.
The extended signal stability of the nanoglo dual luciferase reporter assay enables an easy, addmixread assay protocol. Protect the luciferase substrate assay buffer mixture from light. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. Dual luciferase reporter gene assay kit is used for sequential measuring the. Both words are derived from the latin word lucifer meaning lightbearer. Unlike the chip or emsa assays, which only assess the ability of a protein to interact with a region of dna, a luciferase assay is able to establish a functional. When luciferin is added to the luciferase sample, there is an immediate flash of light that reaches peak intensity within 0. However, the oneglo ex reagent uses a new assay chemistry to increase the stability of both the luminescence signal and the reconstituted reagent. The light then begins to decay rapidly with a half life around 0.
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