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