Promega luciferase reporter assay protocol

A protocol for reporter quantitation in mammalian cells. Our luminescence assay offering includes reporter gene as well as atpmonitoring assays. In this dual reporter system, both firefly photinus pyralis and renilla reniformis sea pansy luciferase activities are measured in succession within the same well as both are functional enzymes. 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. 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. For users working with transformed cells, a cell lysis buffer will be needed for. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. Luciferase assay system perelman school of medicine at. For assays in the gloresponse tm 9xgal4uasluc2p, either the pbinder. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Reporter assay and dualluciferaser promega corporation. Microplate luminometer has a preprogrammed protocol for quick setup of the dualluciferase.

Luciferase assay system quick protocol fb026pdf 102 kb english. The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. The assay begins when 2 luciferase expressing plasmids one of inducible and the other of constitutive expression are transfected into hek293 cells. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. The pgl2 luciferase reporter vectors are designed for the quantitative analysis of factors that potentially regulate mammalian gene expression. This system requires cell lysis prior to performing the assay and. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Consecutive detection of firefly and renilla luciferase activities in a single sample. Promegas dual luciferase reporter assay biocompare product.

Typically, a promoter region containing 2 to 3 kb of a 5. Reagents can be used with injectionbased protocols or with cell lysates made with passive lysis buffer. Both words are derived from the latin word lucifer meaning lightbringer. Dual luciferase reporter system promega 24 well plate transfections cells should be transfected with a test reporter firefly luciferase and a normalization reporter driven by a constitutive promoter renilla luciferase prltk thymidine kinase promoter works. The system, provided with cell culture lysis reagent cclr, also can be used for reporter quantitation. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 telephone 6082744330 fax 6082772516 printed in usa.

It has broad applications across various fields of cell and molecular biology wherever you want to measure or track expression of a cloned gene. In vitro transcription and capping of gaussia luciferase mrna followed by hela cell transfection duration. Higher sensitivity, longlived luminescence, more storage options. Dualluciferase reporter assay system protocol promega. Luciferase assay systems quick protocol card, fb026 promega. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate a stabilized luminescent signal. I performed a dual luciferase assay using pgl3 firefly luc as experimental reporter and prltk renilla luc as internal control promega assay kit.

Information on related products, including the luciferase assay system, is provided in sections 5, 6 and 7. Luciferase reporter assay system 1,000 assays e4550 each system contains sufficient reagents for 1,000 standard assays. Cell culture lysis reagent and reporter lysis buffer may be used with the luciferase assay system for reporter quantitation in mammalian cells. In this protocol, cells transfected with a luciferase reporter plasmid are lysed. Dualluciferase reporter assay system technical manualpdf. Protocol for dna transfection and luciferase assay using. Improved dualluciferase reporter assays for nuclear receptors. Robust firefly luciferase assay with improved performance for high and ultrahighthroughput applications. 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 genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. Promega luciferase assay systems luciferase assay w. Luciferase reporter assay kit university of michigan. In a pinch, all three of the dual kits can be used to measure firefly luminescence, but the second reporter must match the kit. Figure 1 illustrates the two enzymatic reactions, which take place sequentially within the same assay well.

The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. A sensitive reporter assay for continuousprocessing on robotic systems. Nanoglo dualluciferase reporter assay protocol promega. 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. Such cellbased assay data taken together with computational target predictions or expression and proteomics data provides strong evidence that a particular 3utr is directly regulated by the mirna of interest. Luciferase assays allow for the study of transcriptional gene expression, virus life cycles, and cell viability, making them significant tools for drug development in reporter gene, cytotoxicity, and cell proliferation applications. The nanoglo dualluciferase reporter nanodlr assay system allows sensitive detection of firefly and nanoluc luciferases. The luciferase reporter assay kit is suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter. Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. A protocol for measuring firefly and renilla luciferases sequentially from a single sample. Video describing the use of the dualluciferase reporter assay system. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a. Dualluciferase reporter assay system harvard university. The convenient addmixmeasure protocol generates both firefly and renilla luciferase luminescence signals from cells that have not been preconditioned or.

The renilla luciferase assay system is formulated with a. The luciferase assay system is a homogeneous assay system. Luciferase assay system protocol promega corporation. Dualluciferase reporter dlr assay on the spectramax i3x. Protocol for dna transfection and luciferase assay using pfrwt plasmid. Description antibodydependent cellmediated phagocytosis adcp is an important mechanism of action moa of therapeutic antibodies designed to recognize and mediate the elimination of virusinfected or diseased. 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. Renilla luciferase assay system is designed to provide a fast, sensitive method of detecting luciferase from sea pansy renilla reniformis. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. Some luminometers directly inject the reagents into the cell lysate. Description continued the adcc reporter bioassay, complete kit raji, contains all the components and reagents necessary to perform.

The luciferase protocol described utilizes the dualglo luciferase assay system from promega which is designed for high throughput assays. Nanoglo dualluciferase reporter assay nanodlr system. 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. Promega offers the pgl4 series of firefly and renilla luciferase vectors designed. For kits e1500, e1501, e4030, e4530 and e4550, prepare luciferase assay reagent lar by. L 1 vial luciferase assay substrate lyophilized l 105ml luciferase assay buffer l 1 protocol promega corporation. The dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Visit the web site to verify that you are using the. Introduction firefly luciferase is widely used as a reporter for studying gene regulation and function, and for pharmaceutical screening 1, 2. Promega also offers a set of reporters containing the luciferase gene from the sea. Dualluciferase reporter assay system promega corporation. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Luciferase reporter assay in drosophila and mammalian. Problem with luciferase reporter assay researchgate.