Bioluminescent Reporter Assays_Overview
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Bioluminescent Reporter Assays_Overview - 1

Reporter Assays for versatile applications Gene Regulation | Cell Signaling Pathways | HTS Screens | RNA Interference Protein Stability | Protein:Protein Interaction | Receptor Interaction Biosensors | Bioassays | Bioluminescent Imaging

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The Brightest Luciferase Reporter Smartphone image of a 384-well plate with NanoLuc®-expressing cells.

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Supporting science around the world With a portfolio of more than 3,000 products covering the fields of genomics, protein analysis/expression, cellular analysis, drug discovery, and genetic identity, Promega is a global leader in providing innovative solutions and technical support to scientists in academic, industrial and governmental settings. Promega provides products globally through its 15 branches and over 50 distributors. Serving more than 100 countries, Promega supports laboratories with its molecular tools, technical support, and customer service. Promega was first certified to ISO...

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Table of Contents 1 Introduction Bioluminescent Reporter Assays 6 1.2 NanoLuc® - Renilla - Firefly : A Comparion of Luciferases 7 1.3 NanoLuc® - The Brightest Luciferase Reporter 8 1.4 Experimental Design of Reporter Assays 9 2.1 Promoter Studies with Dual-Luciferase Reporter Assays 12 2.2 Signal Transduction - Analysis of Signaling Pathways using Luciferase Reporters 14 2.3 Compound HTS - Coincidence Luciferase Reporters 22 2.5 Protein Stability Sensors Based on NanoLuc® Luciferase 28 2.6 Protein:Protein Interaction (PPI) Assays -...

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6 Dual Assays for the Quantification of Two Luciferases in a Single Sample 56 6.1 Dual Assays Combining NanoLuc® and Firefly Luciferase 58 6.2 Dual Assays Combining Firefly and Renilla Luciferase 60 7 Live-Cell Substrates for Dynamic Real-Time Measurements & Imaging 62 7.1 Live-Cell Substrates for the Detection of NanoLuc® Luciferase 63 7.2 Live-Cell Substrates for the Detection of Firefly Luciferase 65 7.3 Live-Cell Substrates for the Detection of Renillla Luciferase 66 8 Detection with GloMax® Luminometers 68 9.4 NanoBRET™ Protein:Protein...

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Luciferase Reporter Assays 6 NanoLuc®- Renilla - Firefly: A Comparision of Luciferases 7 NanoLuc® - The Brightest Luciferase Reporter 8 Experimental Design of Reporter Assays 9 1.1 Luciferase Reporter Assays Luciferases are widely used as reporter genes across various fields of cell biology, including gene expression at the transcriptional, translational, and posttranslational level as well as the generation of biosensors and protein interactions in live cells. The term luciferase refers to a class of enzymes catalyzing a light-emmitting reaction by oxidation of their respective...

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1.2 NanoLuc® – Renilla – Firefly: A Comparison of Luciferases Luciferases are well suited for setting up highly sensitive and easy-to-quantify reporter assays. Traditionally, a combination of firefly and Renilla luciferase is applied to study gene regulation. The firefly luciferase (Fluc) originates from the American beetle Photinus pyralis, while Renilla luciferase (Rluc) derives from the sea pansy Renilla reniformis. By contrast, the newly developed and highly optimized NanoLuc® luciferase (Nluc) originates from the deep-sea shrimp Oplophorus gracilirostris. Its markedly increased signal...

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1.3 NanoLuc® – The Brightest Luciferase Reporter NanoLuc® luciferase (Nluc) is a genetically optimized luciferase that has been developed by directed enzyme evolution of a luciferase from the deep-sea shrimp Oplophorus gracilirostris. Nluc catalyzes an ATP-independent conversion of its optimized substrate, furimazine, yielding luminescence 150-times brighter than FLuc or RLuc (Fig. 1.2). Furthermore, the Nluc substrate furimazine displays enhanced stability with a half-life greater than two hours and a lower background activity, which both open up a new field for bioluminescence imaging....

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1.4 Experimental Design of Reporter Assays The experimental design is critical to the success of any reporter assay. Generally, it can be divided into the following key steps: 1 cloning of reporter vector constructs, 2 transfection & cultivation of cells, and 3 signal development & detection (Fig. 1.3). Usually, the most time consuming step is the generation of a reporter construct whose respective design depends on the particular question to be addressed (Fig. 1.4). Studies on transcriptional regulation – e.g. promoter studies – require the insertion of a regulatory DNA sequence into a...

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2.1 Promoter Studies with Dual-Luciferase Reporter Assays 12 2.2 Signal Transduction - Analysis of Signaling Pathways using Luciferase Reporters 14 Cellular Stress Response 19 2.3 Compound HTS - Coincidence Luciferase Reporters 22 Measure miRNA Activity with pmir Reporter Vectors 26 Measure siRNA Activity with psiCHECK™-2 Reporter Vector 27 2.5 Protein Stability Sensors Based on NanoLuc® Luciferase 28 NanoBRET™ Target Engagement 34 2.8 Protein Tagging and Detection System - NanoBiT® HiBiT 36 2.9 Live-Cell Biosensors for cAMP, cGMP or Protease Activity -...

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Applications of Luciferase Reporters Introduction – Applications of Luciferase Reporters Luciferase-based detection systems are broadly applied by scientist from academia as well as pharmaceutical industries due to its unparalleled sensitivity, dynamic range, versatility and ease of use. Starting from the 1990s, Promega has been continuously developing luciferase-based products and technologies. The development of the highly effective NanoLuc® Luciferase (Nluc) opened up novel exciting possibilities and improvements to luminescence applications, including monitoring of protein stability and...

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Applications of Luciferase Reporters 2.1 Promoter Studies with Dual-Luciferase Reporter Assays Application Studies on promoter activity, promoter deletion analysis, or analysis of single nucleotide polymorphisms (SNPs) within promoter sequences. Promoter deletion analysis Promoter region Description One common application of luciferase reporter genes is analyz- Regulating region ing the function of cis-acting genetic elements such as promot- ers (“promoter bashing”). Typically, deletions are made within a promoter region, and their effects on coupled expression of a luciferase reporter gene...

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