Egfr is the receptor for members of the egf family and is a transmembrane glycoprotein that has tyrosine kinase. I cloned the promoter into a pgl3 plasmid and co transfesfected this promoter plasmid with a renilla vector in hek293t cells to assess transfection efficiency. Characterization of chemical libraries for luciferase. The assay consists of a genetically engineered jurkat t cell line that expresses a luciferase reporter driven by an nfatresponse element nfatre. Adpglo kinase assay application notes tyrosine kinase series. Flexstation 3 luciferase assay setup parameters figure 2. Riiia receptor, v158 high affinity variant, and an nfat response element driving expression of. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. The biolux gluc flex assay kit includes a higher concentration coelenterazine substrate and an additional stabilizer. In this assay, two enzymatically different luciferases are coexpressed and their distinct luminescent actitivies are measured sequentially after cell lysis in a relatively simple singletube format.
Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. Product components and storage conditions product size cat. Introduction firefly beetle photinus pyralis luciferase is one of the most popular reporter. Dual luciferase assay data analysis issues protocol online. Characterization of chemical libraries for luciferase inhibitory activity douglas s. In flex mode, flexstation 3 microplate reader was set to read luminescence from the bottom of the plate. Measuring the dualluciferase reporter assay on the glomax. Luciferase assay, alternative extractions freeze and thaw. Including coenzyme a in the reaction enhances the sensitivity of the assay and provides a sustained light reaction.
Luciferase reporter gene assay, high sensitivity sigmaaldrich. The luciferase assay system was developed for reporter quantitation in mammalian cells. The realtimeglo mt cell viability assay determines the number of viable cells in culture by measuring the reducing potential of cells and thus metabolism mt. The dualluciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Highthroughput functional screening using a homemade dual. The luminescent signal positively correlates with adp amount fig. Furthermore, the integrated format of the dlr assay provides. The homogeneous protocol makes the assay amenable to automation in 96 and 384well formats. Detecting ubiquitination of target proteins in live cells. The tcrcd3 effector cells nfat are provided in a thawanduse format as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell propagation 3. Grow li cells transformed with pfrwt plasmid dna in appropriate volume of lb or 2xyt media with ampicillin overnight and isolate dna. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. Dualluciferase reporter assay system harvard university. This listings of restriction sites for the pgl2 luciferase reporter vectors are provided in sections 7.
Since a single reagent lyses cells and contains all of the components required to generate a luminescent signal that is proportional to the. These performance characteristics make the bioassay. Transfer the bioglo luciferase assay buffer into the amber bottle containing the bioglo luciferase assay substrate, and mix by inversion until the substrate is thoroughly dissolved. Moreover the bioassay can be performed in a single day. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube.
Application of the dualluciferase reporter assay to the. Austin, and james inglese, nih chemical genomics center, national human genome research institute, national institutes of health, bethesda, maryland 208923370. To mimic assay conditions, such as an inhibitory dose response of a compound, gaussia lysate was diluted experimental reporter and added 1. Equilibrate the bioglo luciferase assay buffer to ambient temperature, protected from light. Nih3t3 cells expressing the luciferase gene from rous sarcoma virus were lysed with 1x cell culture lysis reagent 48. An extremely sensitive reagent for rapid quantitation of firefly luciferase. Figure 4 that the rlu increases slightly as the luciferase reaction continues, but the increase is the same for all. Review and cite luciferase assay protocol, troubleshooting and other. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 6082744330 fax 6082772516 5. The pgl2 luciferase reporter vectors are designed for the quantitative analysis of factors that potentially regulate mammalian gene expression. 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.
Highthroughput functional screening using a homemade dualglow luciferase assay. Do not store luciferase assay reagent with dry ice. Cells and pdl1 aapcchok1 cells provided in a frozen, thawanduse format, and 3 an optimized protocol, that together yield a quantitative bioassay that exhibits low variability and high accuracy. Luciferase reporter assay kit university of michigan. Bioglo luciferase assay system bioglonl luciferase. A split luciferase complementation assay to study proteinprotein interactions within arabidopsis protoplasts in 96well plates is described in this protocol. The assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinasesmaking it ideal for both primary. Reporter assay and dualluciferaser promega corporation.
Oz biosciences luciferase assay kit instruction manual. These factors may be cisacting, such as promoters and enhancers, or transacting, such as various dnabinding factors. The oneglo luciferase assay system is designed for use with the following culture media containing 010% serum. Typically, a reporter gene is cloned with a dna sequence of interest into an expression.
Protocol for dna transfection and luciferase assay using pfrwt plasmid. Thus, the dlr assay system allows accurate, reproducible quantitation of very low levels of renilla luciferase activities expressed in prepared cell lysates. Protocol for dna transfection and luciferase assay using. Our automated transfection protocol is readily adaptable to. The introduction of the dual luciferase assay in mid1990 was a major breakthrough 8,9. Assay lysates for luciferase activity and protein concentration. I was wondering whether it is possible to use a luciferase assay to do so. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. The luminescent signal is proportional to the amount of lactate in the sample and. An external file that holds a picture, illustration, etc. 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.
Luciferase assay system quick protocol fb026pdf 102 kb english. It has broad applications across various fields of cell and molecular biology wherever you want to measure or track expression of a cloned. Adcc reporter bioassay, core kit technical manual tm383. Luciferase assays and biotek synergy microplate reader. Nih3t3 cells expressing the luciferase gene from rous sarcoma virus were lysed with 1x cell culture lysis. Adpglo kinase assay is a luminescent kinase assay that measures adp formed from a reaction. Enzymatic steady state between deacetylase, developer enzyme and luciferase is typically achieved within 20 minutes with a signal halflife of 6090 minutes after steady state is achieved. For an overview and information on the development of the dualglo luciferase assay system, see sections 6. Adpglo kinase assay is a luminescent kinase assay that measures adp formed from a kinase reaction. The alphasynuclein promoter is first cloned into a reporter plasmid containing the firefly. A typical dual spectral luciferase reporter assay protocol is summarized below figure 6. The assay combines a simple, addmixread format, effector cells provided in frozen, thawanduse format, and an optimized protocol to provide a bioassay that has low variability and high accuracy. The assay combines a simple, addmixread format, effector cells provided in a frozen, thawanduse format, and an optimized protocol for a bioassay that has low variability and high accuracy.
The assay consists of adcc bioassay effector cells which are jurkat t cells engineered to express fc. Thaw luciferase assay reagent at temperatures below 25c and mix well before use. I could make a construct with a tandem duplication of the enhancer element, insert into a plasmid and see if there is any. Hdac 2mediated signal is persistent in a purified biochemical assay panel a and in a cellbased lytic assay panel b. The luciferin is then utilized in a firefly luciferase reaction to generate light figure 3. The luciferase assay kit is intended for the detection of the expression of firefly luciferase and is not suitable for. The lightswitch luciferase assay system is a fully optimized reporter system. To see articles that cite the use of the dualglo luciferase assay system, visit. Adp is converted into atp, which is converted into light by ultraglo luciferase fig. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. Secreted dual reporter assay with gaussia luciferase and. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Dualluciferase assay performed in a 96well white walled, clear bottom plate.
Nih3t3 cells expressing the luciferase gene from rous sarcoma virus were lysed with 1x cell culture lysis reagent 48 hours after. Equilibrate all reagents to room temperature before starting the assay. 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. Adcc reporter bioassay, complete kit raji technical. After following the protocol for the dualglo luciferase assay system and assaying luminescence, i normalized the data to empty vector control as recommended by promega. 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. Protocols for restriction digestion, alkaline phosphatase treatment, linker ligation and transformation of competent cells can be found in reference 2. Technical bulletin pgpglo assay systems instructions for use of products. Eukaryotic cell lysis this protocol is optimized for use with eukaryotic cell cultures. The assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinasesmaking it.
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