The celltiterglo luminescent cell viability assay kit uses luciferase as the. However, i am faced with a choice for my firefly and renilla luciferase reporter assays. Normalized firefly luciferase activity firefly luciferase activityrenillaluciferase activity for each construct was compared to that of the pmirglo vector noinsert control. The nano glo 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. Nanoglo dualluciferase reporter assay protocol promega. Nanoglo dualluciferase reporter assay system injectors. Due to their increased rate of degradation, these destabilized reporters respond faster and often display a greater magnitude of response to rapid transcriptional events. Dualluciferase reporter assay system protocol promega. The bright glo luciferase assay system is designed for use with the following culture media containing 010% serum. 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 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 dual glo 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. Dualluciferase reporter assay system harvard university. Transient transfection an overview sciencedirect topics.
The oneglo ex assay provides high sensitivity with an approximately 2 hour luminescence half life with increased reconstituted reagent stability. The assay begins when 2 luciferase expressing plasmids one of inducible and the other of constitutive expression are transfected into hek293 cells. The cells were incubated for 8 h with a transfection mixture containing 500 ng of firefly luciferase containing reporter plasmids pgrp78pro160luc or flagtagged xbp1luc and 1 ng of renilla luciferase containing plasmid phrlcmv promega as an internal control. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or. Bright glo luciferase assay system is designed for continuousprocess systems. Dualglo luciferase assay system promega corporation. Rluc uses coelenterazine as a substrate, but nanoluc luciferase is a furimazine substrate. They are incredibly versatile, and can be designed to measure virtually any cellular or. In dualglo luciferase assay protocol, firefly luciferase is measured and quenched, followed by renilla luciferase, in the same well. Dualglo luciferase assay system from promega biocompare. May 26, 2010 transfected cells were assayed 2024 hours post treatment with dualglo luciferase assay system promega, madison wi.
Assays in 96wells plates were read with the turner biosystems sunnyvale, ca glomax 96 microplate luminometer, 0. In vitro transcription and capping of gaussia luciferase mrna followed by hela cell transfection duration. The oneglo ex luciferase assay system quantitates firefly luciferase expression in multiwell plates using a simple addmixread protocol. After following the directions for reagent preparation by promega. Apr 05, 2019 this all comes down to substrate specificity. Nanoglo dualluciferase reporter assay system promega. The dualglo luciferase assay system is designed to allow highthroughput analysis of mammalian cells containing genes for firefly and renilla luciferases.
Brightglo luciferase assay system protocol promega. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have. The nanoglo dualluciferase reporter assay system opens new research to the use of bioluminescence in a format you already know. The oneglo luciferase assay system employs new assay chemistry to provide a robust, homogeneous assay. The robust, homogeneous assay chemistry gives high assay sensitivity and has a 30minute signal half life. The extended dynamic range of the glomax discover system allows you to easily measure various sample signal intensities on the same plate over a sevenlog range in luciferase concentration. Twentyfour hours after transfection, cells were induced for 6 hours by the addition of 1. 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. Do i use the dualluciferase reporter assay system or. Interleukin6 il6 is a major mediator of the acute phase response apr that regulates the transcription of acute phase proteins apps in the live. Jan 24, 2007 the promega dualglo luciferase assay system is a kit which offers the possibility of quantifying both firefly and renilla gene expression in mammalian cells in a few steps and in sequence. Optimizing dualglo luciferase assays with the synergyht. Luciferase reporter assay for deciphering gpcr pathways. Either measuring substrate consumption or overall production, the enzyme assays are used to monitor enzymatic activity.
The dualglo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. Consecutive detection of firefly and renilla luciferase activities in a single sample. Renilla luciferase assay system is designed to provide a fast, sensitive method of detecting luciferase from sea pansy renilla reniformis. Based on promising dual glo cell data on spectamax m5e, future experiments are planned to investigate the utility of flexstation 3 fluidics to enable flash dual luciferase cell based assays in 384well format. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or prelysed. Calculate relative luciferase activity by dividing results from the luciferase assay over the renilla.
The nano glo luciferase assay system provides a simple, singleaddition reagent that generates a glowtype signal in the presence of nanoluc luciferase with a halflife of approximately 120 minutes in commonly used tissue culture media. Dualglo luciferase assay system technical manualpdf. Measuring the dualglo luciferase assay on the glomax. Incubate the plate at room temperature for 30 minutes to lyse cells and quench the nanoluc signal. System uses the activities of luminescent proteins. I am working on dual luciferase assays using dualglo kit promega. Because of this, a fireflyrluc system should be measured using the dualglo luciferase assay system e2920 or dualluciferase reporter assay system e1910. The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. So far ive only seen people using promega glo buffer in. This article describes the protocol for measuring luminescence using the glomax discover system with the dualglo luciferase assay system. The firefly luciferase reagent is first added to the cells in medium directly. 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.
To see articles that cite the use of the dual glo luciferase assay system, visit. This article provides information on when to use firefly and when to use nanoluc luciferase as a primary reporter, and how both reporters can be used together to measure two biological responses in the same sample. With this approach, the two luciferase activities can be exploited to monitor reporter gene activity and normalize gene activity in a single sample. To see articles that cite the use of the dualglo luciferase assay system, visit. Dualglo luciferase assay system quick protocol fbpdf. Dualglo luciferase assay system protocol promega corporation. Dualglo luciferase assay system is designed for use in mammalian cell culture. 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. For an overview and information on the development of the dual glo luciferase assay system, see sections 6. The oxidative stress response in caenorhabditis elegans. I performed a dual luciferase assay using pgl3 firefly luc as experimental reporter and prltk. Performing most dualluciferase assays involves adding two reagents to each sample and measuring luminescence following each addition. Review and cite dual luciferase assay protocol, troubleshooting and other.
Each transfection was done in triplicate and samples were then averaged. Creb1 is affected by the micrornas mir223p, mir26a5p. The signal halflife under these conditions will generally exceed 25 minutes at 22c and is independent of enzyme concentration where the luciferase concentration is mar 25, 2015 in vitro transcription and capping of gaussia luciferase mrna followed by hela cell transfection duration. The nano glo dual luciferase reporter nanodlr assay system allows sensitive detection of firefly and nanoluc luciferases. Aliquots of varying volumes can be stored, and the number of freezethaw cycles should be noted. Changes in luciferase gene expression are measured using dual glo luciferase assay system promega, madison, wi which includes the use of two reporters, firefly and renilla luciferases. Nanoglo luciferase assay system, promega, promega vwr. Transfect cells with pgl34 plasmid and prl vector 50. Email promega technical services if you have questions on use of this system. Robust firefly luciferase assay with improved performance for high and ultrahighthroughput applications. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a single sample. For each transfection, luciferase activity was averaged.
Transcriptional regulation of retinol binding protein 4 by. The bioglo luciferase assay system provides a highly sensitive, robust, homogeneous reagent, the bioglo assay reagent with a new luciferase substrate, for the detection of firefly luciferase reporter gene expression in the adcc reporter bioassay. This convenient addandread system generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or prelysed. Potent inhibition of firefly luciferase coupled with the highintensity luminescence of nanoluc luciferase maximizes sensitivity for detection of both. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate a. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. 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. Conditions affecting luciferase reagents and assay.
The nanoglo dualluciferase 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. The system is a convenient alternative to firefly photinus pyralis reporter systems and is designed to yield reliable, linear results for a concentration range over 7 orders of magnitude. The nanoglo dualluciferase reporter nanodlr assay system allows sensitive detection of firefly and nanoluc luciferases. The assay uses a twostep, additiononly process to make these measurements in a single well of a plate, negating the need to run parallel assays. Improved dualluciferase reporter assays for nuclear receptors. Presence of metabolically active cells1 is signaled by atp quantitation. Nanobret crbn and vhl ternary complex assays technical. Hidden camera investigation on what really happens to your car cbc marketplace duration. The dual glo luciferase reagent is sensitive to freezing and thawing, with about a 15% loss in firefly relative light units after 5 freezethaw cycles promega. The dual glo 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. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. The brightglo luciferase assay system is designed for use with the following culture media containing 010% serum. 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.
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. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. In the nanodlr assay protocol, firefly luciferase activity is measured first using the oneglo ex luciferase assay reagent. Dualglo luciferase assay system e2940 from promega. Assays in 384well plates were read with the thermo fisher scientific. The dual glo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. Luciferase reporter assay in drosophila and mammalian tissue. 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. After the 30minute incubation is complete, measure total luminescence on a luminometer.
With an injecting luminometer, it couldnt be simpler. Dual luciferase enzyme standards assay results at lower concentrations in 384well plates. Cell lysates were collected after 48 hr transfection and luciferase assays were performed using the promega madison, pa dual glo luciferase assay system e2920 according to the manufacturers protocol. The renillaglo luciferase assay system is a singleaddition reagent that generates a glowtype signal with renilla luciferase. Cellular assays have always been a powerful tool in the research lab. Glo, format is amenable to assays or screens in 96, 384 or 1,536well plates. Luminescent signals generated in the dual luciferase reporter assay system by firefly and renilla. In the dual glo luciferase assay, the activity of the primary reporter firefly luciferase is correlated with the effect of specific stimuli, and the. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12well dish. The topflash assay is a luciferase reporter assay, and it can be used to monitor the concentration of both wnt and rspondin in conditioned media. Promega dual luciferase reporter dlr assay systems the dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. If using the glomax discover system, select the celltiter glo protocol. Promega has developed destabilized luciferase reporters by genetically fusing a protein degradation sequence to the luciferase genes. Product components and storage conditions product size cat.
817 219 1347 994 1412 294 1126 769 207 664 1408 775 665 636 1099 989 1508 250 1099 921 1267 856 940 1053 503 289 122 931