Gaussia activity after adding gluc flex assay solution to the sample. An extremely sensitive reagent for rapid quantitation of firefly luciferase. Use biorad assay for protein concentrations in 20ul of. Typically, a promoter region containing 2 to 3 kb of a 5.
Protocol for dna transfection and luciferase assay using pfr. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. After quantifying the firefly luminescence, this reaction is quenched, and the renilla luciferase reaction is simultaneously initiated by adding. Use a standard protocol to transfect mammalian cells with a firefly luciferase plasmid. The dualglo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. This firefly luciferase assay kit is designed for simple and efficient quantitation of firefly luciferase reporter enzyme activity from cultured cells with high sensitivity and linearity. Note the luciferase substrateassay buffer mixture is best if used within one month when stored at 20c or within one year when stored at 70c.
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. Typically, a reporter gene is cloned with a dna sequence of interest into an. Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a single sample. Dualglo luciferase assay system quick protocol fb pdf 125 kb english. Luciferases are widelyused reporters of biological activity, due to their bright signals and nearly nonexistent background luminescence. Grow cells and assay them directly within the same multiwell plate. Thaw luciferase assay reagent at temperatures below 25c and mix well before use. Luciferase assays cell viability assay kits biotium. Four different mammalian cell types were cotransfected with firefly and renilla luciferase. Luciferase assay protocol reading luciferase and renilla signal using promega dual luciferase reporter assay note. Luciferase assay system with reporter lysis buffer 100 assays e4030 luciferase assay reagent, 10pack 1,000 assays e1501 luciferase assay system freezer pack 1,000 assays e4530 luciferase assay system 1,000 assays e4550 luciferase assay reagent 1,000 assays e1483.
In addition, the luciferase reporter vectors contain numerous features aiding in the structural characterization of the putative regulatory sequences under investigation. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. Luciferase as a reporter of gene activity in plants. Introduction firefly beetle photinus pyralis luciferase is one of the most popular reporter. Product components and storage conditions product size cat.
In dualglo luciferase assay protocol, firefly luciferase is measured and quenched, followed by renilla luciferase, in the same well. The pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. The bioglonl 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. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. Luciferase assay system protocol promega corporation. The assay of this genetic reporter is rapid, sensitive and quantitative. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. 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. Cells were harvested 48 h post transfection and subjected to the luciferase assay following muthumanis protocol with.
Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12well dish. Bioglo assay reagent is functionally tested for performance in the adcc reporter bioassay and is intended for use with this or other bioassays. Thaw firefly luciferase assay buffer at room temperature. A typical dual spectral luciferase reporter assay protocol is summarized below figure 6. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Allow the luciferase substrateassay buffer mixture to reach room temperature. This lysis buffer is compatible with reagents for assaying the activity of gaussia as well as other luciferases e. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube.
Performing the nanoglo dualluciferase reporter assay 3. Do not store luciferase assay reagent with dry ice. No need to remove culture medium or wash cells prior to adding assay reagent. Preparation of the nanoglo luciferase assay reagent remove the nanoglo luciferase assay substrate from storage and mix by pipetting. I could make a construct with a tandem duplication of the enhancer element, insert into a plasmid and see if there is any. Renilla luciferase assay system is designed to provide a fast, sensitive method of detecting luciferase from sea pansy renilla reniformis. Summary of changes the following change was made to the 615 revision of this document. Ideal for high and ultrahighthroughput applications. Development of a cytotoxicity assay based on transient expression of different luciferases in cytosol. Dualluciferase reporter assay system harvard university. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. Luciferase reporter assay kit university of michigan. Assay lysates for luciferase activity and protein concentration. Reporter gene assays several commercially luciferase reporter assay kits are available that contain an expression vector containing the luciferase gene.
This kit is a new and improved replacement for the firefly luciferase assay kit catalog no. For manual luminometers, the substrate solution is mixed by hand with the. The following reagents are supplied with this product. Dual luciferase reporter dlr assay certification bmg labtech. 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. Nanoglo dualluciferase reporter assay nanodlr system. Luciferase reactions were performed according to manufacturers instructions using titration curves of purified recombinant quantilum recombinant firefly luciferase promega or purified recombinant renilla luciferase ray biotech. The extended signal stability of the nanoglo dualluciferase reporter assay enables an easy, addmixread assay protocol. I was wondering whether it is possible to use a luciferase assay to do so. The luciferase assay system yields linear results over at least eight orders of magnitude.
Dispense 100l of the luciferase assay reagent into luminometer tubes, one tube per sample. The luciferase assay system is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Brightglo luciferase assay system is designed for continuousprocess systems. Luciferase assay system 100 assays e1500 each system contains sufficient reagents for 100 standard assays. The robust, homogeneous assay chemistry gives high assay sensitivity and has a 30minute signal halflife. 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. Luciferase assay system quick protocol fb026pdf 102 kb english. 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. 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.
The oneglo luciferase assay system employs new assay chemistry to provide a robust, homogeneous assay. Luciferase enzymes were diluted in 1x passive lysis buffer from the assay kit tested. Luciferase assays and biotek synergy microplate reader. 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. Renilla luciferase assay system promega corporation.
To mimic assay conditions, such as an inhibitory dose response of a compound, gaussia lysate was diluted experimental reporter and added 1. The renilla luciferase assay system is formulated with a. Dualluciferaser reporter assay system technical manual tm040. Brightglo luciferase assay system is designed specifically to meet the needs of continuousprocess systems by providing robust, homogeneous assay chemistry that achieves high assay. The dlr assay is a two step reaction that uses two luciferase enzymes, fire. The luciferase assay system was developed for reporter quantitation in mammalian cells. The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. Thus, the dlr assay system allows accurate, reproducible quantitation of very low levels of renilla luciferase activities expressed in prepared cell lysates. To see articles that cite the use of the dualglo luciferase assay system, visit. Nanoglo luciferase assay system technical manual, tm369. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum.
Passive lysis buffer using either the passive or active lysis procedure. Extra lysis buffer for use with firefly luciferase assay kits, only required if using well sizes larger than 24well. Genecopoeias secretepair system offers the unique advantage of a secreted dualreporter luminescence system, which allows reporter assays in live cells. A luciferase reporter assay is usually performed to investigate the strength and regulation of a promoter. Assay protocol 6 references 9 ordering information 10 instruction manual 3 introduction firefly luciferase is widely used as a reporter for studying gene regulation and function, and for pharmaceutical screening1,2. It has broad applications across various fields of cell and molecular biology wherever you want to measure or track. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Thaw the nanoglo luciferase assay buffer if stored at 20c but do not exceed 25c. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or.
Genecopoeias secretepair system offers the unique advantage of a secreted dualreporter luminescence system, which allows reporter assays in. Pdf luciferase as a reporter of gene activity in plants. Protocol for dna transfection and luciferase assay using pfrwt plasmid. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a. 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. The dualluciferase reporter assay or dlr is widely used to study gene transcription and regulation.
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. Performing most dualluciferase assays involves adding two reagents to each sample and measuring luminescence following each addition. A protocol for reporter quantitation in mammalian cells. Nanoglo dualluciferase reporter assay system nextgen.
Reagents can be used with injectionbased protocols or with cell lysates made with passive lysis buffer. The oneglo luciferase assay system provides a sensitive, robust, homogeneous assay for detection of firefly luciferase reporter gene expression in mammalian cells. The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. It is a very sensitive genetic reporter due to the lack. 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.
Extending the properties of oneglo reagent, oneglo ex reagent employs a new assay chemistry to increase both the stability of the luminescence signal and. Performing most dual luciferase assays involves adding two reagents to each sample and measuring luminescence following each addition. The gluc flex assay solution was prepared as suggested in the protocol. The luciferase assay system is substantially improved over conventional assay. Pdf an improved singlestep lysis protocol to measure luciferase. Highthroughput quantitation of firefly photinus pyralis luciferase expression in mammalian cells requires highly sensitive reagents that can adapt to continuousprocess robotic systems. Prepare treatment medias, thaw cms depending on the assay 2. Development and characterization of a novel luciferase based. Transfect cells with pgl34 plasmid and prl vector 50.
The dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Development and characterization of a novel luciferase. Protect the luciferase substrateassay buffer mixture from light. 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.
Including coenzyme a in the reaction enhances the sensitivity of the assay and provides a sustained light reaction. 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. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Standard luciferase aassy protocol for manual luminometer. Allow cells to incubate in treatment media for 30 min at 37c 5 % co2. The renillaglo luciferase assay system is a singleaddition reagent that generates a glowtype signal with renilla luciferase. Luciferase reporter assay for unlocking ligandmediated. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. Here we share a multiplex assay protocol that allows sequential measurement of cell viability cell number and luciferase activity of the same sample in a multiwell. Prepare the luciferase reagents in the interim, following promega instructions. Luciferase assay substrate and dissolve the substrate in the assay buffer with the help of a pipette to make working assay solution. Luciferase cell lysis buffer lclb is a proprietary formulation developed to produce mammalian cell lysates for reporter assays. Pdf multiplexing fluorogenic esterasebased viability assay. Moreover, the manufacturers protocol recommended that the ratio of renilla luciferase expression vector.
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