Luciferase assay reagent by combining one volume of nanoglo luciferase assay. Dualluciferaser reporter assay system technical manual tm040. The renilla luciferase assay system is designed to provide a fast and sensitive method of detecting sea pansy renilla reniformis luciferase. A commonly used reporter gene is the luciferase gene from the firefly photinus pyralis. A pipeline to process dual luciferase assay results scienceparkstudygroupdualluciferase. All of the luciferase vectors contain the panomics signature sequence. This system is a convenient alternative to firefly photinus pyralis reporter systems and is designed to yield reliable, linear results for a concentration range over seven orders of magnitude.
Dualluciferase reporter assay system harvard university. Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors. Promega offers the pgl4 series of firefly and renilla luciferase vectors designed for use with the dlr assay systems. Chapter 23 detection of proteinprotein interactions in. The dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Luciferase mrna transfection of antigen presenting cells. The experimental setup was similar for each assay combination.
It is a very sensitive genetic reporter due to the lack of any endogenous activity in mammalian cells or tissues3, 4. Recombinant renilla luciferase fused with his tag at cterminal was expressed in e. Biologically relevant assays for oncology biological industries. Promega s multiplexed luciferase reporter and cell viability. The system contains sufficient reagents to perform 1,000 luciferase assays 100l per assay. Dualluciferase reporter assay system promega corporation. Dualluciferase reporter assay system technical manualpdf 771 kb.
Has anybody used the oneglo luciferase assay from promega. 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. An assay reagent for the detection of luciferase which comprises luciferin and a thiol reagent selected from the group consisting of a noncoa thiol reagent, and coa, wherein the assay reagent is free of luciferase. A luciferasefluorescent reporter influenza virus for live. In a method for detecting the presence of a beetle luciferase in a sample suspected of containing the luciferase, comprising. 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. Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. Measuring luminescence of the brightglotm luciferase assay. If this occurs, dilute your sample with 1x lysis buffer and repeat the assay. The celltiterglo assay generates a glowtype luminescent signal, produced by the luciferase reaction, which has a halflife of greater than five hours. Luciferase assay system, 10pack e1501 from promega.
Luciferase assay reagent by combining one volume of nanoglo. Assay lysates for luciferase activity and protein concentration. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube. Feb 01, 1994 similar to the complex of coabeetle luciferase excited oxyluciferin, luciferylcoa occurs in any solution in which luciferin, atp, coa and a beetle luciferase, in an enzymatically active form, are together, including in vitro e. However, once this reagent has been added, how long you wait to measure the signal becomes a. Sep 01, 2006 such systems have found widespread application through the availability of commercial assay kits the dual luciferase reporter assay system, cat. They can be performed easily by adding a reagent that provides components necessary to generate a luminescent signal directly to cells or a cell lysate. The reagent according to claim 1 which additionally comprises adenosine triphosphate. To measure the detection limit of the luciferase assay, we measured the luciferase activity of purified parasites. In a white flatbottom 96well microplate for luciferase assays, mix 25 l of luciferase assay solution with 10 to 25 l of the collected tissue culture supernatant samples. Any search on dual luciferase reporters usually generates a lot of returns. The bioluminescent signal can be detected and quantified using the bioglo luciferase assay system, also included in the kit, and a standard luminometer such as the glomax discover system. Lipidbased reagents are especially popular because they combine efficient.
The oneglo luciferase assay buffer and oneglo luciferase assay substrate, provided with this system, are combined to form the oneglo reagent see section 3. Product components and storage conditions product size cat. This extended halflife eliminates the need for reagent injectors and provides flexibility for continuous or batchmode processing of multiple plates. I am a beginner and i find abnormally huge variations of the dual luciferase assay ratios between duplicate samples. Less than 10 20 moles of luciferase have been detected under optimal conditions. Promega steadyglo luciferase assay system fisher scientific. Celltiterglo luminescent cell viability assay protocol. The dual luciferase reporter dlr assay system provides an efficient means of performing two reporter assays.
Less than 1020 moles of luciferase have been detected under optimal conditions. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a single sample. To see articles that cite the use of the dualglo luciferase assay system, visit. Luciferase reporter assay kit university of michigan. I am carrying out the dual luciferase assay promega on different cell lines. The efficiency of the luciferase ivt rna assay was thus further confirmed by a direct comparison with the 51 crrelease assay using either k562a2 cells or primary dcs as target cells. Multiplexing luciferase reporter and cell viability assays. Signal detection may become saturated when measuring very high lightemitting samples in a luminometer or scintillation counter. For users working with transformed cells, a cell lysis buffer will be needed for. The luciferase assay system was developed for reporter quantitation in mammalian cells.
These vect ors are intended for resear ch use only and should not be used commercially. Nanoglor luciferase assay system technical manual tm369. An extremely sensitive reagent for rapid quantitation of firefly luciferase. At promega, our favorite kind of light in nature is bioluminescence. Western blot molecular weight standards biorad laboratories. Can be used for assays in singletube luminometers or in. The dualluciferase reporter dlr assay system provides an efficient. Dot blotting, immunogen, protein array, function assay, etc. Protocol for use with manual or singleinjector luminometers. Bioluminescent reporter assays are an excellent choice for analyzing gene regulation. Using the renilla lucifearse assay kit promega, the measured net relative light unit rlu was approximately.
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. Promega corporation 2800 woods hollow road madison, wi 537115399 usa. 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. Ecl western assay kit amersham biosciences, piscataway, nj. 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. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a. E1910, promega, madison, wi, usa and alternative protocols based on the same principles. 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. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. I am a beginner and i find abnormally huge variations of the dual luciferase assay ratios between duplicate samples or even the repeated assay on the same sample immediately. The oneglo luciferase assay system employs new assay chemistry to provide a robust, homogeneous assay. 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. Format of the dualluciferase reporter assay in 96well plates.
Promega luciferase assay systems fisher scientific. Prepare luciferase assay reagent ii resuspend a bottle of lyophilized luciferase assay substrate luciferin in luciferase assay buffer and label larii stable for one year at 80. Antigoat antibody conjugated to horseradish peroxidase emd biosciences, san diego, ca. The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. Use biorad assay for protein concentrations in 20ul of. This system is a convenient alternative to firefly photinus pyralis reporter systems and is designed to yield reliable, linear results for a concentration range over seven orders of.
As part of my job i occasionally search the literature for papers citing use of promega products in new or interesting ways. The luciferase assay system is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Resuspend lyophilized luciferase assay substrate in 10 ml of luciferase assay buffer ii and label larii stable for one year at 80. Bmg labtechs highend multimode plate reader pherastar is used to perform multiplexed luciferase reporter and cell viability assays. The pd 1pd l1 blockade bioassay includes the necessary medium and serum to thaw, plate and assay the cells. 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. Prepare the luciferase assay solution see the table of materials by diluting the nluc substrate 1. Promega offers the pgl4 series of firefly and renilla luciferase vectors designed. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. The simple addmixread assay format and optimized reagents combine to create. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Promegas luminescent celltiterglo assay 2 was multiplexed with either the luminescent enduren live cell substrate, or the luminescent viviren live cell substrate. 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.
Chapter 23 detection of proteinprotein interactions in live. A protocol for combined photinus and renilla luciferase. For these reasons, luciferase is widely used in the pharmaceutical and biotechnology industries. Promega s multiplexed luciferase reporter and cell. The brightglo luciferase assay system from promega corporation has been developed specifically to maximize the sensitivity of the assay reagent while providing a luminescent signal halflife generally exceeding 25 minutes. A search for dual luciferase on highwire press generates over 700 articles from 2009 alone.
Dualglo luciferase assay system technical manual tm058. Promega introduces a superior dualreporter technology. In vivo refolding assay in vivo chaperone activity in vivo. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll.
C need 100ml per assay, plus about 2ml extra for the tube volume for luminometer. A firefly luciferase and b renilla luciferase signals were. Parasite pellets were lysed in passive lysis buffer promega and used in the luciferase assay according to the instructions provided by the manufacturer of the dualluciferase reporter assay system promega using lumat lb9507. Luciferase assays are useful tools for studying a wide range of biological questions. Never tried the promega dual luciferase assay myself, but know that the renilla luciferase gives a much higher signal than the firefly luciferase. 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. After 24 hr rest, cells were stimulated with lps il10 for 2 hr. The signal you get rlu depends on the assay you are running and the luminometer you are using. The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to luciferase assay reagent ii. The dualglo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning. Phone 6082744330 or 8003569526 fax 6082772601 dualluciferase and dualluciferase assay protocols dualluciferase reporterassay and dualluciferase reporter assay systems instructions for use of products e1910, e1960 and e1980. Sep 15, 2014 luciferase assays are useful tools for studying a wide range of biological questions.
Luciferase reporter vector, therefore the positions indicated on the vector map should be adjusted accordingly. The luciferase assay system yields linear results over at least eight orders of magnitude. This product is available through the promega helix onsite stocking program. Luciferase assay system 100 assays e1500 each system contains sufficient reagents for 100 standard assays. Instructions for use of products e2920, e2940, e2980. The firefly luciferase is a 62,000 dalton protein, which is active as a monomer and does not require subsequent processing for its activity. Dualluciferaser reporter assay system tm046 promega. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. If your signal is to low, you can usually increase it by adjusting the gain of your luminometer. Dualluciferase reporter assay system protocol promega.
1394 1190 855 414 796 1143 1481 1543 1108 548 610 1309 786 146 1469 433 1582 1526 1477 702 1117 423 950 746 420 1356 729 762 640 1057 712 97 1120 84 227 934 226 976 1482 993 107 135