Cell Signaling Dual-Glo® Luciferase Assay System Product Dual-Glo® Luciferase Assay System For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Dual-Glo® Luciferase Assay System is a homogeneous reagent system that enables fast and simple quantitation of a stable luminescent signal from two reporter genes in a single sample. This convenient “add-and-read” system generates both firefly and Renilla luciferase luminescence signals from cells that have not been preconditioned or prelysed. The Dual-Glo® Luciferase Assay System provides high Z´-factors for cell-based, high-throughput screening applications. With the Dual-Glo® System, internal controls can be established to minimize sample variability by reducing false positive and false negative readings caused by nonspecific factors such as cytotoxicity. In the Dual-Glo® Luciferase Assay, the activity of the primary reporter is correlated with the effect of specific stimuli, and the activity of the co-transfected control reporter provides an internal control to normalize results. The system is optimized for batch processing both 96- and 384-well plates and is compatible with a wide variety of mammalian cell culture media. Features: • Increased Precision and Accuracy: Normalize primary reporter results with an internal control, a co-reporter that minimizes effects of cell number and health, transfection efficiency and nonspecific cellular responses. • Homogeneous Format: Perform fewer steps. Assay cells directly in growth medium for both reporters. No centrifugation or lysis step is required. • Stable Signal: Obtain flexibility for either batch or continuous processing of 96- and 384-well plates. Each luminescent signal may be measured for up to 2 hours after reagent addition. • Convenience: Screen efficiently with this simple, two-step assay that is ideal for any luminometer. On-board injectors not required. • Wide Dynamic Range: Analyze high and low reporter activity without sample dilution. Linear over at least 6 logs of enzyme concentration for each reporter. • Choose Your Configuration: Learn more about our custom options for this product at: www.promega.com/myway/ Storage Conditions: Store Dual-Glo® Substrates at –20°C. Store Dual-Glo® Buffers below 25°C. Protocol Dual-Glo® Luciferase Assay System Technical Manual Dual-Luciferase® Reporter Assay System Product Dual-Luciferase® Reporter Assay System Dual-Luciferase® Reporter Assay System 10-Pack Dual-Luciferase® Reporter 1000 Assay System Available Separately Passive Lysis 5X Buffer For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Dual-Luciferase® Reporter (DLR™) Assay System provides an efficient means of performing two reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis or sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a luminescent signal lasting at least one minute. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated simultaneously by adding Stop & Glo® Reagent to the same sample. Both assays can be completed in about 4 seconds using a Size Cat.# Price (Fr) 100 assays E1910 376.00 1,000 assays E1960 3290.00 1,000 assays E1980 2820.00 30 ml E1941 150.00 Part# TM058 0 None Protein Method of Normalization Effect of transfection conditions on reporter results analyzed using different normalization methods. HEK 293 cells were transfected with pGL4.13[luc2/SV40] expressing firefly luciferase and pGL4.74[hRluc/TK] expressing Renilla luciferase. Transfections were performed using both optimal and suboptimal lipid:DNA ratios (indicated as Optimal and Suboptimal Transfection conditions). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (Cat.# E1960). Protein concentrations were determined using the Coomassie® Plus BradFord Reagent (Pierce). Firefly luciferase data were either not normalized (None), normalized to total protein (Protein), or normalized to Renilla luciferase activity (Vector). Data represent the average ± standard deviation of triplicate samples and are expressed as a percent of the optimal transfection for each normalization condition. Vector 120 40 80 Size Cat.# Price (Fr) 10 ml E2920 414.00 100 ml E2940 3105.00 10 × 100 ml E2980 Pls. Enq. luminometer with reagent auto-injectors. In the DLR™ Assay System, both reporters yield linear assays with attomole (<10–18) sensitivities and no endogenous activity in the experimental host cells. Furthermore, the integrated format of the DLR™ Assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions. For best results with the Dual-Luciferase® Assay, Promega recommends using a luminometer that has been validated for use with the assay. These luminometers are qualified as DLReady™. For a listing of qualified instruments, please visit the DLReady™ Validated Luminometers page. The pGL4 Luciferase Reporter Vectors are designed for use with the DLR™ Assay Systems. A Renilla luciferase vector with constitutive expression may be used in combination with any experimental firefly luciferase vector to co-transfect mammalian cells. Notice for Cat.# E1960 and E1980: Sufficient Passive Lysis Buffer is provided to perform 1,000 assays with cells grown in 96-well plates (typically 20μl of 1X PLB per well). For applications requiring more lysis reagent (e.g., >100μl/well), additional Passive Lysis Buffer may be purchased separately. Features: • Greater Accuracy: Renilla luciferase internal control allows for more accurate results. • Convenient: Samples don’t have to be split, which saves plates and time. • Sensitive: Allows study of weak promoters, low-level expression/regulation, and expression in cells that transfect poorly. • Linear: Range extends 7 logs; very active samples typically do not need dilution. Storage Conditions: Store at –20°C. Protocol Dual-Luciferase® Reporter Assay System Technical Manual Dual-Luciferase® Reporter 1000 Assay System Technical Manual Part# TM040 TM046 Optimal Transfection Suboptimal Transfection 15 For complete and up-to-date product information visit: www.promega.com/catalog 261 Reporter Assays and Transfection Percent Optimal Transfection 6398MA Pagina 264
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