We established a quantitative reporter gene protocol, the prluc assay system, allowing the sequential measurement of photinus and renilla luciferase activities from the same extract. Use biorad assay for protein concentrations in 20ul of. Jun 24, 1997 the comparison conventional assay, which is an adaptation of the assay published in the first reports of luciferase used as a reporter of genetic events, such as transcription, mol. Dualluciferase reporter assay system measures firefly and renilla luciferases from a single sample. Jan 29, 20 see also promega dualluciferase reporter assay system as well as promega pgl3 luciferase reporter vectors available from promega corporation, madison, wis. 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. The assay couples glucose oxidation and nadh production with a bioluminescent nadh detection system 1,2 figure 1. The webinar also discusses the concept of the coincidence reporter assay, an assay developed at the nih that is especially useful for monitoring reporter. Celltiterglo luminescent cell viability assay protocol. Dualluciferase reporter assay system protocol promega. Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors.
Realtimeglo mt cell viability assaypdf promega corporation. The new assay offers the experimental freedom of the dualglo reporter assay system with regard to signal halflife and the brightness of the dualluciferase reporter assay system. The luciferase assay system yields linear results over at least eight orders of magnitude. Luciferase activity was measured 24 h after transfection using the dualglo luciferase assay system promega. Cell culture lysis reagent and reporter lysis buffer may be used with the luciferase assay system for reporter quantitation in mammalian cells. An extremely sensitive reagent for rapid quantitation of firefly luciferase. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. Quantitation of luminescent signal from each of the luciferase reporter enzymes may be performed immediately following lysate preparation without the need for dividing samples or performing additional treatments. A protocol for combined photinus and renilla luciferase. The signal halflife under these conditions is greater than 5. Caspaseglo 8 assay technical bulletinpdf 1103 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. Hek293 cells were transfected with varying amounts of the pnl1.
Normalization of the data included two sequential steps. This assay is broadly useful for class iii enzymes, but sensitivity and performance will vary with catalytic efficiency of particular isoenzymes. Promega luciferase assay systems print promega luciferase assay systems. E1910, promega, madison, wi, usa and alternative protocols based on the same principles. Ideally suited for high and ultrahighthroughput applications, the oneglo assay contains a new luciferase substrate, resulting in a reagent that is more stable, more tolerant to sample components, and has less odor. Celltiterglo luminescent cell viability assay technical bulletin. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. Antibody profiling by luciferase immunoprecipitation. 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. Luciferase reporter assay using mir507, mir518e or mirlet7a and the nefl 3utr. The oneglo luciferase assay system provides a highly sensitive, robust, homogeneous assay for detection of firefly luciferase reporter gene expression in mammalian cells. Product components and storage conditions product size cat. General considerations the steadyglo luciferase assay system is designed for use with the following culture media containing 010% serum. Instructions for use of products e2920, e2940, e2980.
Data from table 1 are expressed as positive values for upregulation and negative values for downregulation x1. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. This vector is based on promega dual luciferase technology, with firefly luciferase luc2 used as the primary reporter to monitor mirna regulation and renilla luciferase hrlucneo acting as a control reporter for normalization and selection. Email promega technical services if you have questions on use of this system. Comprehensive luciferasebased reporter gene assay reveals.
Nanoglo dualluciferase reporter assay nanodlr system. A firefly luciferase and b renilla luciferase signals were. Caspaseglo 8 assay is a homogeneous luminescent assay that measures caspase8. Less than 10 20 moles of luciferase have been detected under optimal conditions. Nanoglo luciferase assay system provides a simple, singleaddition reagent that generates a glowtype signal in the presence of nanoluc luciferase. Protocol for dna transfection and luciferase assay using pfr. The luciferase assay system is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Ideally suited for high and ultrahighthroughput applications, the oneglo assay contains a new luciferase substrate, resulting in a reagent that is more stable, more. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Reporters provide a highly sensitive, quantifiable metric for cellular events such as gene expression, protein function and signal transduction. When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to luciferase assay reagent ii.
Luciferase assay to study the activity of a cloned. 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 luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube. Promega e4530 luciferase assay system freezer pack. Prepare treatment medias, thaw cms depending on the assay 2. Madison, wi, usa in a luminometer turner biosystems luminometer, promega according to the manufacturers instructions. It is a very sensitive genetic reporter due to the lack of any endogenous activity in mammalian cells or tissues3, 4.
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 luciferase assay system was developed for reporter quantitation in mammalian cells. The reagent is prepared by mixing nanoglo luciferase assay substrate and nanoglo luciferase assay buffer and contains an integral lysis buffer to use directly on cells expressing nanoluc luciferase or culture medium when the luciferase. Monitoring signal stability of the nanoglo live cell reagent in hek293 cells. Protocol for dna transfection and luciferase assay using. The nanodlr assay reagents should give a signal halflife of approximately 2 hours at 22c for. The oneglo luciferase assay system employs new assay chemistry to provide a robust, homogeneous 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. Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.
Prepare the luciferase reagents in the interim, following promega instructions. Nanoglo dualluciferase reporter assay protocol promega. Allow cells to incubate in treatment media for 30 min at 37c 5 % co2. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Description firefly luciferase is widely used as a reporter for studying gene regulation and function, and for pharmaceutical screening1, 2. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the prluc assay system was formulated under the aspect of full compatibility with standard methods for protein.
After 24 hours, cell culture medium was exchanged for optimem i reduced serum medium, and. Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. The nanoglo dualluciferase reporter nanodlr assay system allows sensitive detection of firefly and nanoluc luciferases. Summary of changes the following changes were made to the 915 version of this document. Antibody profiling by luciferase immunoprecipitation systems. Protocol for dna transfection and luciferase assay using pfrwt plasmid. Promega luciferase assay systems fisher scientific. Systems include stop and glo buffer and substrate, one vial of luciferase assay substrate, 10 ml luciferase assay buffer ii, and 30 ml passive lysis 5x buffer. 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. A new luminescence assay for autoantibodies to mammalian cellprepared insulinomaassociated protein 2. Systems contain one vial each of brightglo luciferase assay substrate and buffer. Luciferase based assays have become an invaluable tool for the analysis of cloned promoter dna fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. The luciferase assay system is substantially improved over conventional assay methods in both sensitivity and simplicity.
The assay can be used with other nonlytic assays from promega 2,3. These target sites can be introduced by cloning putative mirna binding sites alone, or the 3. Dualluciferase reporter assay system harvard university. Luciferase assay system, 10pack e1501 from promega. 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. Data from reporter assays before gray and after the normalization red against the effect of the mirnas on the firefly luciferase reporter alone are shown. Promega luciferase assay system components fisher scientific. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. The homogeneous assay procedure involves adding a single reagent celltiterglo reagent directly to cells cultured in serumsupplemented medium. Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. Luciferase assay to study the activity of a cloned promoter. The luciferase assay system is a homogeneous assay system. Celltiter glo assay is designed for use with multiwellplate formats, making it ideal for.
Celltiterglo one solution assay technical bulletinpdf. Luciferase assay systems instructions for use of products e1483, e1500, e1501, e1531, e4030, e4530 and e4550. Assay lysates for luciferase activity and protein concentration. Nanoglo luciferase assay system technical manualpdf. The pmirglo dualluciferase mirna target expression vector is designed to quantitatively evaluate microrna mirna activity by the insertion of mirna target sites 3.
Luciferase assay system 100 assays e1500 each system contains sufficient reagents for 100 standard assays. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. This readytouse reagent is based on the original celltiterglo luminescent cell viability assay chemistry and eliminates the need to combine buffer with. Promega corporation, which are incorporated by reference in their entirety. The endpoint format of the realtimeglo mt cell viability assay is much more sensitive and rapid compared to currently available viability assays that measure. The celltiterglo assay is designed for use with multiwell plate formats, making it ideal for automated highthroughput screening hts, cell proliferation and cytotoxicity assays. However, several factors may affect the sensitivity and success of the assay including ph, temperature, and substrate concentration. 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. Sep 01, 2006 such systems have found widespread application through the availability of commercial assay kits the dual luciferase reporter assay system, cat. Promega corporation 2800 woods hollow road madison, wi. The behaviours of the transfected cells were evaluated by a fireflyluciferase reporter gene harbouring the promoter region of i.