The milliflex rapid microbiology detection and enumeration system. Highthroughput assays using a luciferaseexpressing replicon. 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. To perform the luciferase assay, 100 l of each plate lysate is added to 360 l of luciferase assay buffer table i in a luminometer vial. The improved reporter assays showed larger assay dynamics in shorter assay time than using regular luciferase. For manual luminometers, the substrate solution is mixed by hand with the.
Use all components of the lightswitch system as recommended to obtain optimal reporter assay results. A, b transient depletion of tip60 using sirna and increase in tert expression was verified in the same set of cells used for luciferase assay in fig 2b. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The mgatp 2converts the luciferin into a form which is capable of being catalytically oxidized by the luciferase in a high quantum yield chemiluminescent reaction, according to the following equation. Indeed, adenosine triphosphate atp is found in all liv ing organisms and is an excellent marker for. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. Chromium 51cr release assay has been the gold standard for quantifying cytolytic activities of cytotoxic t lymphocytes ctls against target cells and this method is still being used in many laboratories. The luciferase assay system is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of.
Introduction atplitetm is an adenosine triphosphate atp monito ring system based on firefly photinus pyralis luciferase. The firefly luciferase assay is extremely sensitive and extends over a linear range covering at least seven. Assay, high sensitivity can be used in manual or automated luminometers, in micro plate or tube. In the presence of coa, the luciferase assay yields stabilized luminescence signals with significantly greater intensities figure 2 than those obtained from the conventional assay chemistry. Briefly, a mouse fibroblast cell line, 3t3, was grown to near confluency in a 60 ml culture dish in dulbeccos modified eagles medium dmem supplemented with 10% serum. Both luciferases are the smallest and brightest luciferases known and wellsuited for reporter assays. Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors. Biovisions luciferase reporter assay kit provides a simple means for detecting luciferase activity in transfected eukaryotic cells. Here we present a versatile solution to this need, utilizing the small and very bright nanoluc luciferase to assess secretion and injection through the type iii secretion system encoded by salmonella pathogenicity island 1. Luciferase reporter assay kit ii renilla promocell. Renilla luciferase assay system quick protocol, 9fb066 promega.
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. Due to a high luciferase activity in the luciferase reagent, the atp bioluminescence assay. The way we measure luciferase is by washing the 96 well plate, removing the luciferase and atp released in the media by dead cells and then solely measuring the luciferase in the cell after adding. Just recently, nanoluc, a new engineered luciferase based on the small subunit of the luciferase from oplophorus gracilirostris was introduced. We will save the remainder of the lysate for western blotting with specific antibodies to the gfp tag. It has broad applications across various fields of cell and molecular biology wherever you want to measure or. 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.
Dualluciferase reporter assay system harvard university. As seen in figure, this assay kit compares well with the promega brightglow assay system for measurement of luciferase activity. Atp bioluminescence assay to quantify cell cytotoxicity bmg. Renilla and firefly luciferase luminescence were compared in renilla luciferase assay reagent rlar or luciferase assay reagent lar, respectively, over a titration range of 1. The reactions are started by the injection of 200 l of the 0. The dual luciferase format is an endpoint assay providing unsurpassed sensitivity, specificity, and signaltonoise ratios. Dispense 100l of the luciferase assay reagent into luminometer tubes, one tube per sample. The mgatp 2 converts the luciferin into a form which is capable of being catalytically oxidized by the luciferase in a high quantum yield chemiluminescent reaction, according to the following equation. Luciferase reporter gene assay the promega luciferase assay system kit and cell extracts were generous gifts of dr. A firefly luciferase dual color bioluminescence reporter. Titration of recombinant firefly luciferase in the firefly luciferase assay. Assay procedure for 96well plates using dualsample injection.
The reaction is catalyzed by the enzyme luciferase obtained from the firefly photinus pyralis. Optimization of the luciferase assay kit may be necessary for use with your equipment or samples. The advantage of luciferasereporters is a very high sn107 and sensitivity. After short incubation 2 min light intensity detection of. Both words are derived from the latin word lucifer meaning lightbringer. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. The accuracy of the data is improved by using a second constitutively expressed renillaluciferase in a dualluciferase assay format, which removes false positives due to cell death from. Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. The assay begins when 2 luciferase expressing plasmids one of inducible and the other of constitutive expression are. Assay lysates for luciferase activity and protein concentration.
Dual luciferase reporter dlr assay certification bmg labtech. Dual luciferase assay principle 1 assays on flexstation 3. Technical manual dual luciferase reporter assay system instructions for use of products e1910 and e1960. How to interpret dual luciferase assay result rlu and. Renilla luciferase assay system has comparable sensitivity to the luciferase assay system firefly luciferase and 100fold greater sensitivity than a published method for assaying renilla luciferase. Less than 10 20 moles of luciferase have been detected under optimal conditions. Like the luciferase from gaussia princeps, this luciferase is secreted into the medium. Each assay includes a pathwayfocused transcription factor reporter and a noninducible negative control, as well as luciferase and gfp positive controls. Luciferase assay, alternative extractions freeze and thaw. Development of a cytotoxicity assay based on transient expression of different luciferases in cytosol. The scientist produces a construct in which the regulatory region of a target gene is fused with the dna coding sequence for luciferase figure 15. Development and characterization of a novel luciferase based. Luciferase assays cell viability assay kits biotium.
Nanoglo dualluciferase reporter assay nanodlr system. Assay principle the reaction is catalyzed by the enzyme luciferase obtained from the firefly photinus pyralis. Luciferase reporter assay kit tests bioassay systems. This kit can be conveniently used for measuring luciferase activity in 96well plates or in preparations of lysed cells.
The luciferase assay system yields linear results over at least eight orders of magnitude. The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. This can be done by linking the promoter of interest to an easily detectable gene, such as the gene for firefly luciferase, which catalyses a. A simple and sensitive method to quantitatively measure the cytolytic effect of tumorspecific t killer cells is highly desirable for basic and clinical studies. The lightswitch luciferase assay system is a fully optimized reporter system that includes goclone constructs utilizing the rensp luciferase gene and lightswitch luciferase assay reagents. Use biorad assay for protein concentrations in 20ul of.
Protocol for dna transfection and luciferase assay using pfr. Luciferase reporter assay system for deciphering gpcr pathways. A fast and sensitive luciferasebased assay for antibody. 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. The reagent consists of two components, a luciferase reagent buffer component a and luciferase reagent substrate component b. This luminescence assay is the alternative to colorimetric, fluorometric and radioisotopic assays for the quantitative evaluation of proliferation and cytotoxicity of cultured. Steadyluc firefly hts assay kit lyophilized 30028l. A separate dna construct encodes the protein hypothesized to affect transcription. 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. Use of atp bioluminescence for rapid detection and enumeration of contaminants.
If this occurs, dilute your sample with 1x lysis buffer and repeat the assay. Detection limit the detection limit for atp, using a bertholdtype microplate luminometer lb96p, is in the range of 1012 m fig. Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The milliflex rapid microbiology detection and enumeration system 103 fig.
Instruction booklet 6016947 atplite 5,000 assay kit. Luciferase assay system firefly luciferase and 100fold greater sensitivity than a published method for assaying renilla luciferase. Protocol for dna transfection and luciferase assay using. Luciferase substrate is added to assay buffer as needed, so signal doesnt drop during kit storage. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube. Picture show the image of the membrane with spots a or peaks in 3 dimensions b re presenting exactly the place on the membrane. Weve made big improvements to our luciferase assay kits, with simplified assay setup, improved working solution stability, and high sensitivity and linearity. The system contains sufficient reagents to perform 1,000 luciferase assays 100l per assay. Luciferase reporter gene assay, high sensitivity sigmaaldrich. Luciferase assay system, 10pack e1501 from promega. Use of a luciferaseexpressing replicon cell line in an hts antiviral assay.
Dualluciferase reporter assay system technical manual. A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene. Typically, a reporter gene is cloned with a dna sequence of interest into an. Use of atp bioluminescence for rapid detection and. Luminescent signals generated in the dualluciferase reporter assay system by firefly and renilla. When using the microplate format, black or white microplates must be used we recommend using black microplates. Luminescent signals generated in the dual luciferase reporter assay system by firefly and renilla. A nanoluc luciferasebased assay enabling the realtime. Figure 2 functional evaluation of malibuglo reagents generated using different marine luciferases.
Firefly photinus pyralis luciferase is a monomeric enzyme of 61 kda that requires no posttranslational modifications for activity. Atp bioluminescence assay to quantify cell cytotoxicity. The onestep luciferase assay system is designed to be used for highthroughput, sensitive quantitation of firefly luciferase activity in mammalian cell culture. Promega luciferase assay systems fisher scientific. Unlike the chip or emsa assays, which only assess the ability of a protein to interact with a region of dna, a luciferase assay is able to establish a functional connection between the presence of the protein and the amount of gene. 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. The bioassay systems luciferase reporter gene assay kit sllu01k quantifies luciferase reporter gene expression in transfected cells recombinant cells.
Dual luciferase assay for secreted luciferases based on. Using a model system of exogenous or endogenous receptors in hek293 cells, we demonstrated that all four major g protein subfamilies and downstream pathways can be studied in one luciferase reporter assay format. Working range the working range of the atp bioluminescence assay kit hs ii is between 105 and 1012 m atp fig. Renilla luciferase from the sea pansy renilla reniformis is a 36kda monomeric enzyme that catalyzes the oxidation of coelenterazine to yield coelenteramide and blue light with a spectral maximum of 480 nm. This format is available for all cignal reporter assays. The main purpose of the reporter gene assay is to investigate the promoter of a gene of interest, i. Performing the nanoglo dual luciferase reporter assay 3. Development and characterization of a novel luciferase. The light emission from the reaction can be recorded using a luminometer. Comparison of the dart and chromaglo luciferase assay systems equal numbers of hek293t cells were transfected with the following plasmids. When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Quantilum luciferase promega was serially diluted in 1x. The dualglo luciferase reagent can be added directly to cells in growth medium without washing or preconditioning.
The dual luciferase fireflyrenilla assay system is designed to be used for highthroughput, rapid quantitation of both firefly and renilla luciferases from a single sample in mammalian cell culture. 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. For measurement of expressed luciferase in vitro, luciferase is first extracted from transfected cells through cell lysis. A simple and sensitive method for measuring tumorspecific. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. A simple and sensitive method for measuring tumorspecific t.
Typically, a promoter region containing 2 to 3 kb of a 5. Here, we present a comprehensive mirna reporter gene assay protocol that includes a rigorous. The nanolucbased secretion assay features a very high signaltonoise ratio and sensitivity down to the nanoliter scale. Lyophilized format for convenient shipping and storage. I performed a dual luciferase assay using pgl3 firefly luc as experimental reporter and prltk renilla luc as internal control promega assay kit. The assay begins when 2 luciferase expressing plasmids one of inducible and the other of constitutive expression are transfected into hek293 cells. Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly.
Grow li cells transformed with pfrwt plasmid dna in appropriate volume of lb or 2xyt media with ampicillin overnight and isolate dna. Renilla and firefly luciferase luminescence were compared in renilla luciferase assay reagent rlar or luciferase assay reagent lar, respectively, over a titration range. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a. The luciferase reporter gene assay, high sensitivity can be performed in all automated or manual luminometers in tube and microplate formates as well as in scintillation counters, or using photographic films. The dual luciferase assay is a fast reaction with 2 injection steps, one for the fire. Signal detection may become saturated when measuring very high lightemitting samples in a luminometer or scintillation counter. 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. 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. Protocol for dna transfection and luciferase assay using pfrwt plasmid.
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