inter-assay variability stemming from inconsistent dye uptake and spontaneous dye leakage over the course of the assay. The Dual-Luciferase Reporter (DLR) Assay System provides an efficient means of performing two reporter assays. Component A and Component B are combined to form a . This format is available for all Cignal Reporter Assays. The firefly luciferase catalyzes the bioluminescent oxidation of luciferin in the presence of ATP, magnesium and oxygen. Our Amplite Renilla Luciferase Reporter Gene Assay Kit is designed to provide a fast and sensitive method to detect the luciferase from sea pansy (Renilla reniformis). The dual-luciferase format is an end-point assay providing unsurpassed sensitivity, specificity, and signal-to-noise ratios. For agonist, fold of induction = firefly RLU induced / firefly RLU uninduced. Prevalence and Structure Activity Relationships of Luciferase Inhibitors The Luciferase Assay System is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Measurement of luciferase assays is typically performed using a plate-based luminometer. In a single-reporter assay or dual-reporter assay using two experimental reporters, a cell health assay will make it easy to understand the data and at the same time, will account for reduced. Renilla Luciferase Assay System is designed to provide a fast, sensitive method of detecting luciferase from sea pansy (Renilla reniformis).The system is a convenient alternative to firefly (Photinus pyralis) reporter systems and is designed to yield reliable, linear results for a concentration range over 7 orders of magnitude.The Renilla Luciferase Assay System is formulated with a . After the addition of a lysis buffer and a substrate, a luminometer quantifies the luciferase activity. Assay normalization using an internal control co-reporter minimizes the effects of variations in cell number and health, transfection efficiency and nonspecific cellular responses. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Our formulation generates a luminescent product that gives strong luminescence upon . The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. In the DLR Assay, the activities of firefly and Renilla luciferases are measured sequentially from a single sample. Transfect cells with pGL3/4 plasmid and pRL vector (50:1) and plate in a 12 well plate. 3 G), suggesting that E2 . Luciferase Reporter Assay. Luciferase Reporter Assays Firefly and renilla luciferase activities, as indicated by relative luminescence units (RLU) were determined using One-Glo or Dual-Glo luciferase assay kits (Promega) according to the manufacturer's instructions. In case of exogenous luciferase reporter assays, minimum of four independent repeats were performed. Linear results are seen over at least eight orders of magnitude of enzyme concentration, and less than 10 -20 moles of luciferase can be measured under optimal conditions. The Luciferase Reporter Gene Assay is based on the bioluminescence generated during the luciferin/luciferase reaction. Thaw out enough Dual Glo Buffer and Stop & Glo Buffer for the assay. Assay Advantages Increased Precision and Accuracy. Publication types Research Support, N.I.H., Extramural MeSH terms Each assay includes a pathway-focused transcription factor reporter and a noninducible negative control, as well as luciferase and GFP positive controls. For the preliminary experiments, different cell lines were used to transfect the reporter plasmid CRE-luc and the internal . Wash wells once with 1 mL D-PBS -/-, aspirate PBS. Luciferases are attractive as assay reporters due to the high signal to background inherent to bioluminescence, largely due to a very low background signal as no excitation light is required to generate a signal ( 7 ). Promega's Luciferase Assay System was developed for reporter quantitation in mammalian cells.The Luciferase Assay System (Cat.# E1500), provided with Cell Culture Lysis Reagent (CCLR), can also be used for reporter quantitation in plant and bacterial cells (see Section III.E); however, the Luciferase Assay System with Reporter Lysis Buffer (Cat.# Because the reporter enzymes themselves (e.g. Can be readily automated on HTS liquid handling systems. Our Amplite Gaussia Luciferase Reporter Gene Assay Kit uses a proprietary luminogenic formulation to quantify luciferase activity in cell medium. BioAssay record AID 669614 submitted by ChEMBL: Inhibition of eIF4E in rabbit reticulocyte cell lysate assessed as inhibition of cap-dependent translation after 90 mins by luciferase reporter gene assay. Can freeze the cells at this point if needed. Dual Glo Reporter Assay System (Promega # E2910) . Dual-Luciferase Reporter Assay System 100 assays $ 235.00 Your price: Log in Sensitive, Rapid and Convenient Dual-Reporter Assay The Dual-Luciferase Reporter (DLR) Assay System provides an efficient means of performing two reporter assays. If your protein activates the expression of the target gene, the amount of signal produced . Our formulation generates a luminescent product that gives strong . Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. When this protein activates transcription, the cell will produce luciferase enzyme. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein.The name was first used by Raphal Dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. Talk to a Scientist Paraj 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. The Glucocorticoid Receptor (GR)-GAL4 Luciferase Reporter Jurkat Cell Line contains an engineered transcription factor stably integrated into the genome of Jurkat cells, which consists of the glucocorticoid receptor ligand binding domain fused to the DNA binding domain of GAL4. The luciferase reporter gene is a useful tool for determining the efficacy of transfection of plasmid DNA and adenovirus-mediated gene transfer in vivo. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg (++), yielding a fluorescent product that can be quantified by measuring the released light. Manufacturer: Promega E1910 View more versions of this product Catalog No. F , G PC3 and 22RV1 cells were treated with NFs-Exo, CAFs-Exo or CAFs-Exo + anti-miR-1290 for 48 h. Western blot analysis of GSK3, -catenin, c-Myc and cyclin D1 expression in PC3 and 22RV1 cells. Phenol red in culture media does not interfere in this assay. Detection Reagent Considerations Multiple assay detection reagents are also available for each reporter. Luminescent reporter gene assays for . luciferase assay 1 pGL3-basic Compatible with routine laboratory and HTS formats: assays can be performed in tubes or microplates, and measured with any luminometer. Chemical screen for MfR activation using luciferase reporter gene assay. Data are presented as means with standard deviations (n = 3) and an asterisk denotes significant increase in Met-SRC . The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to Luciferase Assay Reagent II. The Gluc reporter assay has been shown to be a sensitive reporter in monitoring endoplasmic reticulum stress and the secretory pathway 1,4,18,20, promoter activity 21, small interference RNA . Luciferase reporter assays can be multiplexed with other assays to assess apoptosis, metabolism and more. White bar indicates DMSO vehicle control, light grey bar indicates the positive control (methyl farnesoate), and dark grey bars indicate screened chemicals. Luciferase reporter assay indicated that these mutations greatly decreased the enhancer activity and enhancer activity of mutant E2-3 could no longer be induced by IFN, while the control mutant E2-3 which was mutated in positions other than STAT1 binding motif showed no significant difference with wild-type E2-3 (Fig. Dual luciferase assay was used to determine TOP reporter activity in PC3 cells. How does a luciferase reporter work? Here, we describe a protocol to measure the transcriptional activity of the HH pathway in normal and cancer cells. High sensitivity and wide detection range: detection of as little of 2 fg luciferase. Application. Taken together, these results demonstrate that the stable reporter cells are useful tools for: (1) detection of signaling pathway-specific ligands; (2) identification of novel ligands for specific TFs, and (3) screening for agonists and antagonists of specific ligands/receptors. The Luciferase Reporter 1000 Assay System (Cat.# E4550) was designed to meet the needs of users who perform a large number of assays, particularly in 96- well plates. The Dual-Glo Luciferase Assay System provides high Z-factors for cell-based, high-throughput screening applications. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay. Activity of 5-HT on different response elements based luciferase assay. Promega offers a choice of 3 different luciferase reporters: NanoLuc Luciferase (Nluc,19kDa), Renilla Luciferase (Rluc, 36kDa) and Firefly Luciferase (Fluc, 61kDa) which vary in size, brightness, and protein half-life. Luciferase Reporter Gene Assay (LRA) Characteristics. Materials. Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well Incubate on rocker for at least 10 minutes We have developed many simple add-mix genetic reporter assays that can be used for a variety 5678MA Cytoplasm Nucleus You will need 100 uL per well of each. Cells transfected with luciferase reporter; Tube-based luminometer (GloMax 20/20) Protocol. Statistical significance was determined by comparing means of different groups and conditions using unpaired 2-tailed Student's t test, and one-way ANOVA. [20] [21] Promoter assays [ edit] The luciferase assays are performed using an empirically derived concentration of adenosine 5-triphosphate (2 m M ATP) that yields maximal luciferase activity when assaying Hep G2 cellular extracts ( 7 ). LUCIFERASE REPORTER CANCER CELL LINES FACILITATE CAR-T DEVELOPMENT. A Luciferase Reporter Assay. Luciferase reporter assays are powerful tools to measure the specific ability of a transcription factor to bind to its consensus sequence and to activate transcription of target genes. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12 . The One-Step Luciferase Assay System is designed to be used for high-throughput, sensitive quantitation of firefly luciferase activity in mammalian cell culture. Positive and negative controls were included. 5 Benefits of Cignal Reporter Assays PERFORMANCE: Both the dual-luciferase and GFP reporter systems provide exceptional sensitivity, reproducibility, specificity, and signal to noise ratio VERSATILITY: Can monitor signal transduction pathway activity utilizing the dual-luciferase reporter system in an endpoint format assay, or measure pathway PR-E1910 $331.00 / Each of 1 . A commonly used reporter gene is the luciferase gene from the firefly Photinus pyralis. Page 2 Order online at ww.atcc.org, call 800.638.6597, 703.365.2700, or contact your local distributor. The reconstituted reagent has been optimized to combine cell lysis and detection into one single step. Our formulation generates a luminescent product that gives strong . Gaussia luciferase is a 20kDa protein which catalyzes coelenterazine oxidation by oxygen to produce light. ONE-Glo Luciferase Assay System 10ml 100,00 Your price: Log in Bright, Stable Luminescence Signal for Batch and Continuous Processing Systems The ONE-Glo Luciferase Assay System provides a highly sensitive, robust, homogeneous assay for detection of firefly luciferase reporter gene expression in mammalian cells. This fusion construct activates firefly luciferase expression which is under the control of a multimerized GAL4 . Luciferase signals were analyzed using the Dual-Luciferase Reporter Assay System. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. firefly luciferase) can be direct targets of small molecules and confound the interpretation of HTS data, novel coincidence reporter designs incorporating artifact suppression have been developed. Multiple corrections were made using post-hoc Tukey test, Bonferroni test and Scheffe . (D) RKO Control*1, APCmut*1 and APCmut*2 cells were treated with either vehicle (0.01% DMSO), 1 M or 2 M lovastatin for 72 hours. Firefly luciferase levels were normalized to renilla luciferase levels. The luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions with a Centro LB 960 luminometer (Berthold Technologies, Oak Ridge . Luciferase Assay. Here we applied these improvements to two reporter formats for studying nuclear receptors. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. ** P This Amplite Luciferase Reporter Gene Assay Kit uses a proprietary luminogenic formulation to quantify luciferase activity in live cells and cell extracts. It uses a proprietary luminogenic formulation to quantify Renilla luciferase activity in cell-based assays. Let's find the product that meets your needs. Secreted luciferase assay protocol eliminates cell lysis 2-4 fold higher signal than firefly or Renilla luciferase Single transfection allows multiple time points from same cell Dual, live-cell assay includes SEAP as well as secreted luciferase: monitor two promoters, or use one reporter as a normalization control More Information Applications After harvesting, the cells were lysed in a passive lysis buffer (Promega, Madison, WI, USA). The system contains sufficient reagents to perform 1,000 luciferase assays (100l per assay). Luciferase-based genetic reporter assays provide sensitive methods for assaying gene expression, enabling the accurate quantification of small changes in transcription resulting from subtle changes in biology. Both words are derived from the Latin word lucifer, meaning "lightbearer", which in turn is derived from the Latin . It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. 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