The luciferase reporter assay kit is suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter. We calculated the ratio between firefly and renilla using the relative light units obtained for each well, to control variation in dna concentrations, cell number and transfection efficiency. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. From the luciferase gene from the luciferase reporter high. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube.
Luciferase complementation assays detect proteinprotein interactions within. Biovisions luciferase reporter assay kit provides a simple means for detecting luciferase activity in transfected eukaryotic cells. The accuracy of the data is improved by using a second constitutively expressed renillaluciferase in a dualluciferase assay format, which removes false positives due to cell death from. The dualluciferase reporter dlr assay system provides an efficient. Jan 27, 2015 activity of 5ht on different response elements based luciferase assay. The system also includes synthetic mirna target reporter constructs for use as. 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. After short incubation 2 min light intensity detection of as little of 2 fg luciferase and as few as 4 cells. Using synergy ht multimode microplate reader to run the. Promega luciferase assay systems fisher scientific. The ratio of the firefly and renilla luciferase values between the negative mirna control and its correspondent.
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. Using luciferase assays to study gproteincoupled receptor. Luciferase substrate is added to assay buffer as needed, so signal doesnt drop during kit storage. So, luciferase uses a very energetic process to create light.
All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed. Promega luciferase assay systems print share promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. After using the dual luciferase assay from promega, however, i am getting luminescence values from 300 to 1200 for firefly luciferase and 1530 for renilla luciferase background is 12. When using the microplate format, black or white microplates must be used we recommend using black microplates. Truly quantitative analysis of the firefly luciferase complementation. The substrate for renilla luciferase is contained in the stop and glo reagent, which also inactivates any firefly luciferase activity present. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence. 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. Dualluciferase reporter assay system promega corporation. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. Introducing a new onestep glow assay kit for highthroughput screenings hts.
I would like to knockdown a protein of interest using sirna usually i do double transfection and then. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. It has a cofactor, termed a luciferin, that forms a highly strained complex with oxygen, using an atp molecule to help set everything up. The luciferase reporter gene assay, high sensitivity can be performed in all automated or manual luminometers in tube and microplate formates as well as in scintillation counters, or using photographic films. Lyophilized format for convenient shipping and storage. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer.
Atp or adenosine triphosphate is the primary source of energy for all living organisms, and by all we mean all. Luciferase reporter assay system for deciphering gpcr pathways. I need to see the reduction in renilla level, while firefly is working as control. Which microplate reader for dual luciferase assays should i buy.
The luciferase assay system is a homogeneous assay system. B cbluc twocolor dualluciferase assay protocol is shown in which cell lysis and detection. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Cell are then imaged using fluorescence microscopy and dsred2 protein to identify wells with failed transfections, and subjected to the luciferase assay. For instance, resveratrol8,9 is a potent luciferase inhibitor that complicates the interpretation of data collected from luciferase based reporter gene assays for this compound. Firefly luciferase is a 62kda monomeric protein found in the lightemitting organ, known as lantern, of photinus pyralis.
Use biorad assay for protein concentrations in 20ul of. Dual luciferase assay system for rapid assessment of gene expression in saccharomyces cerevisiae. Dualluciferase reporter assay and dualluciferase reporter assay system quick protocol fb024pdf 73. The name was first used by raphael dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. Cell debris is removed by microcentrifugation and luciferase activity is. The dual luciferase reporter dlr assay system provides an efficient means of performing. The research group of umezawa first published the principle of the lca using. Luciferases are widely used in biotechnology, for microscopy and as reporter genes, for many of the same. In our hands, the detection of renilla luciferase was not as sensitive as firefly luciferase on a molar basis, with a determined detection limit of 6.
The knockdown of 3utrluciferase reporter activity by let7a was measured in ht1080 cells by cotransfecting 100ng of each reporter construct with 20nm let7a mimic or nontargeting control, incubating for 24 hours, then reading the luminescent reporter signal. Nanoglo dualluciferase reporter assay nanodlr system. Summary of changes the following change was made to the 615 revision of this document. Why you dont need to select a wavelength for a luciferase assay. 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. A new reporter system has been developed for quantifying gene expression in the yeast saccharomyces cerevisiae. Assay lysates for luciferase activity and protein concentration.
Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors. Among them, the recently established luciferase complementation imaging lci assay is the simplest and fastest method for demonstrating in vivo proteinprotein interactions. Here, we present a rapid and sensitive assay based on the classical dual luciferase reporter technique which can be. The gene encoding renilla luciferase is fused to a constitutive. Renilla luciferase from the sea pansy renilla reniformis is a 36kda monomeric enzyme that catalyzes the oxidation of coelenterazine to yield coelenteramide and blue light with a spectral maximum of 480 nm. Saldanha, h angelica gualtieri, g ajay thankamony, i ieuan hughes. Matt carter, jennifer shieh, in guide to research techniques in neuroscience second edition, 2015. Scroll through our luciferinluciferase crash course and get the answers to some. Why you dont need to select a wavelength for a luciferase.
A protocol for measuring the activity of firefly photinus pyralis and. The halflife of gaussia in this buffer system is between 2 to 3 hours. Luciferase assay expected range of raw reading values hi there, i am performing luciferase assays with chromaglo luciferase assay system from promega using the mutlilabel counter victor3. The jove video player is compatible with html5 and adobe flash. The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. The luciferase assay system yields linear results over at least eight orders of magnitude. Performing the nanoglo dualluciferase reporter assay 3. Thus, luminescent measurements made after the addition of this reagent are entirely the result of firefly luciferase activity. The stop and glo reagent used in the dualluciferase assay has a reversible affinity to some types of plastics often used in injector systems. Immediately assay or store at 20c for use within 1 month or 80c for longterm storage. Luciferase assay expected range of raw reading values. Luciferase complementation imaging assay in nicotiana. Luciferase assay system 100 assays e1500 each system contains sufficient reagents for 100 standard assays.
Including coenzyme a in the reaction enhances the sensitivity of the assay and provides a sustained light reaction. The kit includes information about kit components, assay type, halflife, and experimen. A transfection of either 293t or chok1 cells with creluc and prlsv40 plasmids followed by stimulation with 10. Novel application of luciferase assay for the in vitro. These results prove that phage display method is powerful in detecting crucial positions.
Simple luciferase assay for signal transduction activity detection. In order to insure that the residual reagent has been removed, it is recommended that the syringe and tubing be filled with 70% ethanol for approximately 30 minutes. Cell culture lysis reagent and reporter lysis buffer may be used with the luciferase assay system for reporter quantitation in mammalian cells. Get in touch with a nearby distributor or sales representative. Examining multiple cellular pathways at once using multiplex. Luciferase reporter assay kit tests bioassay systems. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. Firefly luciferase with atp shown magenta 8 types of luciferases coelenterate luciferase also called as aequorin the balls yellow shows the bound ca in the enzyme the light is the reaction of the ca with the aequorin 9 luciferase genes 10 bacterial luciferase gene vibrio fischeri. Illumination firefly luciferase enhanced assay kit goldbio. Although units of activity have been proposed for the purified enzyme see discussion currently, no units have been assigned to the worthington preparations.
The luciferase assay was performed with aliquots of the same protein extracts used in the gus assay. A typical firefly luciferase assay is then carried out in an assay cuvette. To see articles that cite the use of the dualglo luciferase assay system, visit. We described an assay for egfr transduction activity utilising the luciferase gene under control of the. The luciferase assay system is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Copy citation download citations reprints and permissions. Basic luciferase assay a and luciferase assay modified by the addition of coa b. Characterization of chemical libraries for luciferase inhibitory activity douglas s. The light emission from the reaction can be recorded using a luminometer. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously.
Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Here we describe a method for cotransfection of individual 3utrreporter. The luciferase assay system incorporates coenzyme a coa for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The luminescent reaction is then triggered by an injection of luciferin, and the emitted light is recorded. The improved reporter assays showed larger assay dynamics in shorter assay time than using regular luciferase. In this assay, protein a or protein b is fused with the aminoterminal or carboxylterminal half of luciferase, respectively. Characterization of chemical libraries for luciferase. 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. Assay of luciferase activity is based upon the measurement of light produced. 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. Austin, and james inglese, nih chemical genomics center, national human genome research institute, national institutes of health, bethesda, maryland 208923370. Typically, a reporter gene is cloned with a dna sequence of interest into an expression vector that is then transferred into cells.
The mink lung cells are stably transfected with luciferase cdna driven by a portion of pai1 promoter. Illuminating insights into firefly luciferase and other bioluminescent. A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene. Both words are derived from the latin word lucifer meaning lightbringer. Gusluciferase fusion reporter for plant gene trapping and. Use this table to help determine the best luciferase assay kit for your experimental needs. In the presence of oxygen, luciferase derives energy from atp and converts luciferin into oxyluciferin. 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 atp bioluminescence assay is a common technique used to quantify atp levels and detect living, metabolically active cells. For example, multiply the single reaction volumes by 3. In a pinch, all three of the dual kits can be used to measure firefly luminescence, but the second reporter must match the kit. I have psicheck2 vector which has both renilla and firefly. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. The amount of light produced is proportional to the amount of the luciferase enzyme and the level of expression of the gene of interest if used as a genetic reporter. Raw data from firefly and renilla luciferase luminescence assay were recorded for each well. Dec 05, 2015 novel application of luciferase assay for the in vitro functional assessment of kal1 variants in three females with septooptic dysplasia sod mark j. 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. Using the clarity luminescence microplate reader to perform. Linear results are seen over at least 8 orders of magnitude of enzyme concentration, and the patented technology incorporated into this assay system allows measurement of less than 1020 moles of luciferase under optimal conditions. Luciferase activity was measured using the dual luciferase assay system promega corp.
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. Bacterial luciferase showing alpha and beta peptides. 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. Dualluciferase reporter assay and dualluciferase reporter assay system quick protocol fb024pdf.
Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Luciferase reporter gene assay principle kaboom latam inc. What is the reason for the effect on renilla signal in the. A commonly used reporter gene is the luciferase gene from the firefly photinus pyralis. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase.
We developed a luciferase reporter assay to screen epidermal growth factor receptor egfr. Application of the dualluciferase reporter assay to the. 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 connection between the presence of the protein and the amount of gene. This kit includes a buffer that will prolong your gaussia signal over several hours. This report describes the optimization and evaluation of a simple singlestep lysis protocol to measure luciferase bioluminescence from genetically modified plasmodium falciparum. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. When this oxygenated luciferin breaks, forming carbon dioxide in the process, it leaves behind a highly excited form that then emits the light. The bioassay systems luciferase reporter gene assay kit sllu01k quantifies luciferase reporter gene expression in transfected cells recombinant cells. Prior to luciferase assay, the media was removed and cells were lysed for 30 min in the presence of 26. Note samples may lose activity with every freezethaw cycle. Basically, this assay requires the luciferin compound, which is obtained from glowing organisms like fireflies, and its corresponding catalyst enzyme called luciferase. I ligated my gene of interest into 3utr of renilla gene.
The system relies on two different reporter genes, renilla and firefly luciferase, to evaluate regulated gene expression. The assay of this genetic reporter is rapid, sensitive and quantitative. For measurement of expressed luciferase in vitro, luciferase is first extracted from transfected cells through cell lysis. Twosite expression immunoassay using a firefly luciferase. When the excited molecule relaxes back to its ground state, it emits light at 562 nm with a quantum. In the dlr assay, the activities of firefly and renilla luciferases are. In addition, the luciferase reporter vectors contain numerous features aiding in the structural characterization of the putative regulatory sequences under investigation. Jun 07, 2019 in a firefly, renilla, or nanoluc luciferase assay, light is produced as a byproduct of a reaction in which a luciferase enzyme converts a substrate to a product. 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.
605 1449 671 604 1264 1462 1243 117 326 967 541 114 1366 528 871 564 1051 616 363 1426 398 608 293 1392 302 469 271 1107 223 1016 1261 1018 1056 609 1424 663 406 309 1161