Incubation with 1st ab step 10 was increased to 1h. 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. To view, download, or print the document of interest. To see articles that cite the use of the dualglo luciferase assay system, visit. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. A split luciferase complementation assay to study proteinprotein interactions within arabidopsis protoplasts in 96well plates is described in this protocol. The luciferase kits can also be used to measure luciferase activity in prelysed cells. Highthroughput functional screening using a homemade dual. Dualglo luciferase assay system promega corporation. We will indirectly assess the levels of luciferase by quantitation of its. There is batch to batch variation of lar, so make sure to use the same batch only. 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.
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. Todays protocol will allow us to quantitatively assess the levels of transfected luciferase in the cells. When using the microplate format, black or white microplates must be used we recommend using black microplates. Atp determination kit provides all of the reagen ts needed to perform this luminescence assay. Luciferase assay system 100 assays e1500 each system contains sufficient reagents for 100 standard assays. Being present a electricity, proteins migerate towards the negative anode inside the poly. Taqman microrna assays protocol harvard university. Open to view the document save target as to download a pdf version of the document to a destination that you choose. Antigens are expressed in cos1 cells as recombinant renilla luciferase rucantigen fusions, and crude extracts are obtained and used without purification.
Immediately, remove frozen luciferase assay reagent lar from 80c freezer. Antibody profiling by luciferase immunoprecipitation. Sonication or other methods of cell lysis may reduce the sensitivity of the assay. The luciferase assay system yields linear results over at least eight orders of magnitude. Addition of a single reagent results in cell lysis, cleavage of the substrate and generation of a glowtype luminescent signal. Product components and storage conditions product size cat. 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 is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. 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 whole assay can be performed in 3 to 4 days as shown by the overview in fig. Luciferase based assays have become an invaluable tool for the analysis of cloned promoter dna fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. The split luciferase complementation assay springerlink. Promega luciferase assay systems an extremely sensitive reagent for rapid quantitation of firefly luciferase. Realtime, livecell assays repeatedly monitor over time and generate multiple data points from a single assay well.
Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Luciferase assay to study the activity of a cloned. 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. Transfect cells with pgl34 plasmid and prl vector 50. Protocol for dna transfection and luciferase assay using. Western blot rifkin lab nyu langone medical center.
Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. We illustrate the usefulness of this system for studying both constitutive telomerase and inducible nf. The lips assay is initiated by incubating crude rucantigen extracts with patient sera in microtiter wells. The thermo scientific pierce bca protein assay kit is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. I take the promoter of interest, insert it before luciferase coding sequence, and get. The assay firefly luciferase is a 62,000 dalton protein which is active as a monomer and does not require subsequent processing for its activity. 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. The luciferase assay system is substantially improved over conventional assay methods in both sensitivity and simplicity. Mix 550 ul of luciferase containg sample or cell lysate with 100 ul of the luciferase assay kit and read immediately in the luminometer. Optimization of the luciferase assay kit may be necessary for use. Application the atp bioluminescence assay kit hs ii is used for highly sensitive. Endpoint assays can provide sensitive, highthroughputamenable assay formats for determining cell health. The assay information file aif a pdf file of the taqman copy number assays protocol pn 4397425 a pdf file of the taqman copy number assays quick reference card pn 4397424 a pdf file of the copycaller software user guide pn 44000042 a pdf file of the copycaller software quick reference card pn 44000043.
Prepare the luciferase reagents in the interim, following promega instructions. Cell viability and cytotoxicity assays are based on colorimetric, fluorometric and bioluminescent detection chemistries. Protocol for measuring neutralizing antibodies against hiv. Assay lysates for luciferase activity and protein concentration. Sdspage, with full name of sodium dodecyl sulfate polyacrylamide gel electrophores, is the most widely used technique to separate proteins from complicated samples of mixture, plays key roles in molecular biology and wide range of subfield of biological research. Performing the nanoglo dual luciferase reporter assay 3. Protocol for dna transfection and luciferase assay using pfrwt plasmid. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. All incubations are performed in a humidified 37oc, 5% co 2 incubator unless otherwise specified. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. In this work a protocol to measure intracellular refolding activity of molecular chaperones is presented. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. Problem with luciferase reporter assay i have a problem with luciferaseexpression assay. Promega luciferase assay systems fisher scientific.
All assay reagents should be close to room temperature at the time of assay. Instructions for use of products e2920, e2940, e2980. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. 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. The nab assay in our sample data is a platebased assay that records neutralization in tzmbl cells as a function of a reduction in tatinduced luciferase luc reporter gene expression after a single round of infection montefiori, d.
Nanoglo dualluciferase reporter assay nanodlr system. Each tube contains 1ml, enough for measuring 5 samples. We present a rapid and inexpensive highthroughput screening protocol to identify transcriptional regulators of alphasynuclein, a gene associated with parkinsons disease. However, several factors may affect the sensitivity and success of the assay including ph, temperature, and substrate concentration. Luciferase assaynormalisation apr172012 hi im carrying out an experiment to look at geneexpression event and how this is changed when different concentrations of a. Dualluciferase reporter assay system harvard university. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. Use biorad assay for protein concentrations in 20ul of. Luciferase reporter assay kit university of michigan. We strongly recommend using the cell lysis buffer and protocols supplied with this kit. 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.
Application of the dualluciferase reporter assay to the. 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. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Incubation with 2nd ab step 12 was increased to 30 min. Can be used for assays in singletube luminometers or in multiwellplate luminometers with autoinjectors. All washes, but in step 8, were increased to 5 min. 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.
This video demonstrates the steps involved in performing the lips assay. This report describes the optimization and evaluation of a simple singlestep lysis protocol to measure luciferase bioluminescence from genetically modified plasmodium falciparum. Review and cite luciferase assay protocol, troubleshooting and other methodology information contact experts in luciferase assay to get answers. Both words are derived from the latin word lucifer meaning lightbringer. Problem with luciferase reporter assay researchgate. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube. A protocol for an assay that measures caspase3 and 7 activities in purified enzyme preparations or adherent or suspension cells. Prepare treatment medias, thaw cms depending on the assay 2. Using the format of a 96well flatbottom culture plate as illustrated template a fig. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12well dish.
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