The knockdown of 3utr luciferase reporter activity by let7a was measured in ht1080 cells by cotransfecting 100ng of each reporter construct with 20nm let7a mimic or nontargeting control, incubating for 24 hours, then reading the luminescent reporter signal using lightswitch luciferase assay reagents. This luciferase reporter assay kit is designed for detection and. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12well dish. The onestep luciferase assay system is designed to be used for highthroughput, sensitive quantitation of firefly luciferase activity in mammalian cell culture. Luciferase reporter assay substrate kit firefly ab228530 provides a fast, simple, and homogeneous bioluminescence assay for the quantification of luciferase activity in live cells and cell extracts. 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. For measurement of expressed luciferase in vitro, luciferase is first extracted from transfected cells through cell lysis. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Dualreporter assays enable researchers to obtain additional information from complex systems with minimal effort. To see articles that cite the use of the dualglo luciferase assay system, visit. The knockdown of 3utrluciferase reporter activity by let7a was measured in ht1080 cells by cotransfecting 100ng of each reporter construct with 20nm let7a mimic or nontargeting control, incubating for 24 hours, then reading the luminescent reporter signal.
Measuring firefly and renilla luciferase activities in drosophila. Luciferase reporter assay kit 200 tests bioassay systems. Cell based reporter assay platform indigo biosciences. This animation describes the basic concept of reporter assays using luciferase, and explains how a dualluciferase assay works. The luciferase protocol described utilizes the dualglo luciferase assay system from promega which is designed for high throughput assays. Luciferase reporter assay kit university of michigan. The system, provided with cell culture lysis reagent cclr, also can be used for reporter quantitation.
Get resources and offers direct to your inbox sign up. Protocol for dna transfection and luciferase assay using. Renilla luciferase has been used as a reporter for studying gene regulation and function in vitro and in vivo5,6. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses.
I have a plasmid with my gene promoter of interest linked to luciferase reporter in the pgl3 basic vector, available from addgene. 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. Luciferase reporter assay in drosophila and mammalian. Biovisions luciferase reporter assay kit provides a simple means for detecting luciferase activity in transfected eukaryotic cells. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. 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. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube.
Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. Titration of recombinant firefly luciferase in the firefly luciferase assay. Some luminometers directly inject the reagents into the cell lysate. In the dlr reporter assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. When a transient or stable transfection assay is developed for a promoter. Also there is a recipe for selfmade dualluciferase reporter substrates 100x times. Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The luciferase assay system was developed for reporter quantitation in mammalian cells. Assay lysates for luciferase activity and protein concentration. 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.
Each assay includes a pathwayfocused transcription factor reporter and a noninducible negative control, as well as luciferase and gfp positive controls. 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 immediately after translation wood et al, 1984. For applications requiring more lysis buffer see assay protocol, additional 5x passive lysis. After short incubation 2 min light intensity detection of as little of 2 fg luciferase and as few as 4 cells. The light emission from the reaction can be recorded using a luminometer. The reagent consists of two components, a luciferase reagent buffer component a and luciferase reagent substrate component b. When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. Common uses for plasmids expressing luciferase luciferase reporter assay. Recently, renilla luciferase has been widely used in multiplex transcriptional reporter assays or as a normalizing transfection control for firefly luciferase assay6,7. 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 dualluciferase reporter dlr assay and dualglo assay enable the sequential measurement of both firefly and renillaluciferases from one sample. Bioluminescence assay systems are being used increasingly in biology and medical research laboratories in addition to or as alternatives to fluorescence and chemiluminescence detection strategies. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. When using the microplate format, black or white microplates must be used we recommend using black microplates.
In dualglo luciferase assay protocol, firefly luciferase is measured and quenched, followed by renilla luciferase, in the same well. Which protocol do you follow for the assay or do you use any kit. Luciferase enzymes isolated from different animal species have inherent variability in light emission. Luciferase reporter assay for unlocking ligandmediated. Luciferase assay system protocol promega corporation. The typical luciferase containing plasmid is most often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated regions, or the effect of mutations of these regulatory elements on gene expression. This luciferase reporter assay kit is designed for detection and quantification of the firefly luciferase reporter enzyme from cultured cells in a simple, efficient, and linear fashion figure 2. We demonstrate that normalization against the effect of the.
Bioassay systems luciferase reporter gene assay kit sllu200 quantifies luciferase reporter gene expression in transfected cells recombinant cells. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate a stabilized luminescent signal. I need to express it in hek 293t cells, which are human cells. Hi there, ive never done a luciferase reporter assay before and am also new to dna cloning etc, which is why im very confused. Luciferase reporter assay substrate kit ab228530 abcam. Some of the benefits of using a secreted reporter molecule like metluc is its nolysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell. Renilla luciferase, a monomeric 36,000 dalton protein, catalyzes. The pgl2 luciferase reporter vectors are designed for the quantitative analysis of factors that potentially regulate mammalian gene expression.
Luciferase reporter gene assay, high sensitivity y version 20. Readytoglow secreted luciferase reporter assay protocol. Readytoglow secreted luciferase reporter assay protocolat. Performing most dualluciferase assays involves adding two reagents to each sample and measuring luminescence following each addition. Our reporter cell systems utilize firefly luciferase reporter gene technology to provide optimal assay sensitivity and dynamic range when quantifying nuclear receptor activity. The typical luciferasecontaining plasmid is most often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated regions, or the effect of mutations of these regulatory elements on gene expression. Gene promoter luciferase reporter assay help protocol online. The accuracy of firefly luciferase reporter can be improved by normalizing to a control reporter, such as renilla luciferase reporter, in the same sample. Reporter assay and dualluciferaser promega corporation. For a general introduction to the theory of the readytoglow secreted luciferase reporter system, please refer to. The dual luciferase format is an endpoint assay providing unsurpassed sensitivity, specificity, and signaltonoise ratios. Dualluciferase reporter dlr assay system provides an efficient means of performing dual reporter assays.
Protocol for dna transfection and luciferase assay using pfr. Furthermore, for cells that are not easily transfectable, it is not practical for luciferase reporters to be introduced by transient transfection as part of the assay protocol. 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. Dual luciferase reporter dlr assay certification bmg labtech. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Hidden camera investigation on what really happens to your car cbc marketplace duration. The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays.
Gaussia luciferase reporter assay for monitoring biological. The system, provided with cell culture lysis reagent cclr, also can be used for reporter quantitation in plant and bacterial cells. Luciferase reporter assay in drosophila and mammalian tissue. Luciferase assay was performed with dualluciferase reporter assay system kit. This assay is based on firefly luciferase, a monomeric 61 kd enzyme that catalyses a twostep oxidation of luciferin, which yields light at 560 nm. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. Transfect cells with pgl34 plasmid and prl vector 50. A recombinant luciferase was serially diluted in 1x firefly lysis buffer with 1 mgml bsa and measured in the assay. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. Dualluciferase reporter assay system technical manual pdf. The system contains sufficient reagents to perform 1,000 luciferase assays 100l per assay.
Luciferase reportersreadytoglow secreted luciferase assay. Bioluminescent firefly luciferase assays sigmaaldrich. Combined with choices for traditional firefly and renilla luciferase assay kits to provide compatibility with multiple systems, as well as superior quality luciferase reporter clones for mirna targets, promoters, and transcriptional response elements, genecopoeias luciferase products provide you with a wealth of tools for luminescence reporter. B the firefly luciferase hts assay is a steadyglow high sensitivity firefly luciferase reporter gene assay kit for the quantification of firefly. The dualluciferase format is an endpoint assay providing unsurpassed sensitivity, specificity, and signaltonoise ratios. Described here is a protocol for detecting the recently established naturally secreted gaussia luciferase gluc in cultured cells as well as in. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Oct 22, 2014 this animation describes the basic concept of reporter assays using luciferase, and explains how a dual luciferase assay works. Dualluciferase reporter assay system harvard university. Use biorad assay for protein concentrations in 20ul of.