Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. It has broad applications across various fields of cell and molecular biology wherever you want to measure or. All washes, but in step 8, were increased to 5 min. 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.
This luciferase does not require atp and catalyzes the oxidation of its luciferin substrate in a photochemical reaction. Protocol for dna transfection and luciferase assay using. This report describes the optimization and evaluation of a simple singlestep lysis protocol to measure luciferase bioluminescence from genetically modified plasmodium falciparum. All incubations are performed in a humidified 37oc, 5% co 2 incubator unless otherwise specified.
The biolux cypridina luciferase assay kit contains the reagents necessary for assaying cypridina luciferase activity. Hp printers cannot print pdfs from adobe reader windows hp. Todays protocol will allow us to quantitatively assess the levels of transfected luciferase in the cells. Luciferase assay system 100 assays e1500 each system contains sufficient reagents for 100 standard assays. To view or download the pdf version of this document, select ftp about 1636 kb.
To save a pdf on your workstation for viewing or printing. 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. Pdf file for file transfer protocol ibm knowledge center. Incubation with 2nd ab step 12 was increased to 30 min. C2bbe1 bcrp knockout and wild type cell lines 24 well assay ready plates catalog number mtox1002pc24 store at room temperature technical bulletin product description compozr zinc finger nuclease zfn technology is a fast and reliable way to manipulate the genome in a targeted fashion. The scientist transfects a cell culture system, such. 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. When using the microplate format, black or white microplates must be used we recommend using black microplates. Protocol for measuring neutralizing antibodies against hiv1. The luciferase assay system was developed for reporter quantitation in mammalian cells. How to split a pdf file adobe acrobat dczelfstudies. 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. The luciferase assay system is substantially improved over conventional assay methods in both sensitivity and simplicity. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity.
Promega luciferase assay systems fisher scientific. A split luciferase complementation assay to study proteinprotein interactions within arabidopsis protoplasts in 96well plates is described in this protocol. 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. Nanoglo dualluciferase reporter assay nanodlr system. 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. Immediately, remove frozen luciferase assay reagent lar from 80c freezer. The luciferase assay kit is the most sensitive analytical tools for measuring gene expression. The name was first used by raphael dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. The firefly luciferase is a 62,000 dalton protein, which is active as a monomer and does not require subsequent processing for its activity. Dual luciferase gene reporter assays to study mirna. We present a rapid and inexpensive highthroughput screening protocol to identify transcriptional regulators of alphasynuclein, a gene associated with parkinsons disease.
Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. Review and cite luciferase assay protocol, troubleshooting and other methodology information contact experts in luciferase assay to get answers. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12well dish. Two proteins of interest, a bait and prey, which are genetically fused to amino and carboxyterminal fragments of renilla luciferase, are transiently expressed in protoplasts.
In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly. Prepare treatment medias, thaw cms depending on the assay 2. The substrate for cypridina is different than coelenterazine, which. The standard luciferase reporter assay protocol for p.
The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. An improved singlestep lysis protocol to measure luciferase. 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. Problem with luciferase reporter assay i have a problem with luciferaseexpression assay. The luciferase assay system yields linear results over at least eight orders of magnitude. The luciferase kits can also be used to measure luciferase activity in prelysed cells. The firefly can convert luciferin to oxyluciferin in the presence of the enzyme luciferase to emit light.
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. It is a very sensitive genetic reporter due to the lack of any endogenous activity in mammalian cells or tissues3, 4. Click here to download a zip file of all pdf files for exchange server protocol documents. Protocol for dna transfection and luciferase assay using pfr. C2bbe1 bcrp knockout and wild type cell lines mtox1002pc24. Protocol to prevent, suppress and punish trafficking in. Protocol for dna transfection and luciferase assay using pfrwt plasmid. The reagent can be added directly to cells in growth medium without washing, and induces cell lysis and acts as a substrate for firefly luciferase, producing a luminescent signal that can be read over a period of two hours. The split luciferase complementation assay springerlink. Description firefly luciferase is widely used as a reporter for studying gene regulation and function, and for pharmaceutical screening1, 2. 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 luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. What is a luciferase assay and what is it useful for.
Luciferase assay systems instructions for use of products e1483, e1500, e1501, e1531, e4030, e4530 and e4550. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. 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 see section 5. I take the promoter of interest, insert it before luciferase coding sequence, and get. Dualluciferase reporter assay system harvard university. Both words are derived from the latin word lucifer meaning lightbringer.
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. Western blot rifkin lab nyu langone medical center. Less than 10 20 moles of luciferase have been detected under optimal conditions. The nanodlr assay reagents should give a signal halflife of approximately 2 hours at 22c for. 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. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. 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. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. All assay reagents should be close to room temperature at the time of assay. Promega luciferase assay systems print share promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Including coenzyme a in the reaction enhances the sensitivity of the assay and provides a sustained light reaction.
A protocol for analysis of mammalian cells containing firefly and renilla luciferase genes, grown in multiwell plates. Transfect cells with pgl34 plasmid and prl vector 50. Luciferase reporter assay kit university of michigan. Why you dont need to select a wavelength for a luciferase. How to make your pdfs open with adobe reader on windows 8. This protocol is easily modifiable to target other genes of interest and may also be adapted for any application requiring highthroughput transfections, such as lentivirus production 22. The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to luciferase assay reagent ii. Disclosures this work was supported by royalties obtained by licensing bacmamreagents us patent 5,731,182. Cypridina is a new secreted luciferase reporter from the ostracod cypridina noctiluca. Print a different pdf file to determine if the issue occurs with a specific pdf file or all pdf files. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. To mimic assay conditions, such as an inhibitory dose response of a compound, gaussia lysate was diluted experimental reporter and added 1. Uses html5 to display pdf files directly in the browser. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses.
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. Dual luciferase gene reporter assays to study mirna function. There is batch to batch variation of lar, so make sure to use the same batch only. The oneglo luciferase assay buffer and oneglo luciferase assay substrate, provided with this system.
Each tube contains 1ml, enough for measuring 5 samples. The amount of light produced is proportional to the amount of the luciferase enzyme and the level of expression of the gene of interest if used as a genetic reporter. We will indirectly assess the levels of luciferase by quantitation of its. Assay lysates for luciferase activity and protein concentration. A typical dual spectral luciferase reporter assay protocol is summarized below figure 6. Click on associate a file type or protocol with a program. Problem with luciferase reporter assay researchgate. When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Allow cells to incubate in treatment media for 30 min at 37c 5 % co2. Prepare the luciferase reagents in the interim, following promega instructions. Fast and easy, accurate and linear, this kit detects and quantify firefly luciferase in transfected eukaryotic cells reaching high sensitivity level. A luciferase assay takes advantage of the innate bioluminescent properties some organisms exhibit, most notably the firefly. In this work a protocol to measure intracellular refolding activity of molecular chaperones is presented.
Using the format of a 96well flatbottom culture plate as illustrated template a fig. Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors. Luciferase assays and biotek synergy microplate reader. The behaviours of the transfected cells were evaluated by a fireflyluciferase reporter gene harbouring the promoter region of i. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube. If adobe reader is installed on your computer, but your pdf files are not opening with. A separate dna construct encodes the protein hypothesized to affect transcription.
Use biorad assay for protein concentrations in 20ul of. The most common scientific assays utilizing luciferase are reporter assays in which transcriptional activation. Evaluation of parameters for a simplified singlelysis step luciferase reporter assay. This protocol supplements the united nations convention against transnational. Protocol for measuring neutralizing antibodies against hiv. Incubation with 1st ab step 10 was increased to 1h. The whole assay can be performed in 3 to 4 days as shown by the overview in fig. Problem with luciferase reporter assay i have a problem with luciferase expression assay. A protocol for combined photinus and renilla luciferase. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific mirna using a luciferase reporter gene. Highthroughput functional screening using a homemade dual. Product components and storage conditions product size cat.
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