Luciferase Reporter Gene Assay Kit *Bright Glow*
OverviewCommon reporter genes include beta-galactosidase, beta-glucuronidase and luciferase. The advantages of a luciferase assay are the high sensitivity, the absence of luciferase activity inside most of the cell types, the wide dynamic range, rapidity and low cost. The most versatile and common reporter gene is the luciferase of the North American firefly Photinus pyralis. The protein requires no posttranslational modification for enzyme activity. It is not even toxic in high concentration (in vivo) and can be used in pro- and eukaryotic cells. The firefly luciferase catalyzes the bioluminescent oxidation of luciferin in the presence of ATP, magnesium and oxygen. This Amplite™ Luciferase Reporter Gene Assay Kit uses a proprietary luminogenic formulation to quantify luciferase activity in live cells and cell extracts. Our formulation generates a luminescent product that gives strong luminescence upon interaction with luciferase. The kit provides all the essential components with our optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments. It has extremely high sensitivity, and can be used for the assays that require demanding sensitivity.PlatformLuminescence microplate readerRecommended plateSolid whiteComponentsCat.#12518Cat.#12519Cat.#12520Component A: Luciferase Sensor (Light-sensitive)1 bottle1 bottle1 bottleComponent B: Assay Buffer1 bottle (10 mL)1 bottle (100 mL)1 bottle (1000 mL)Example protocolAt a glanceProtocol summaryPrepare cells (samples) with test compounds (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)Add equal volume of Luciferase Sensor working solutionIncubate at room temperature for 10 - 20 minutesMonitor luminescence intensity at 560 nmImportant notesThaw all the kit components to room temperature before use. For all luminescent experiments, it is recommended to use white plates to get the best results.Preparation of working solution1. For Cat.# 12518, transfer the whole content of Reaction Buffer (Component B) into the bottle of Luciferase Sensor (Component A) and mix well to make Luciferase Sensor working solution. 2. For Cat.# 12519, add 10 mL of Reaction Buffer (Component B) and for Cat.# 12520, add 100 mL of Reaction Buffer (Component B) into the bottle of Luciferase Sensor (Component A) and mix well. Then, transfer the resulted solution back to the bottle of Reaction Buffer (Component B). Multiple washes are necessary to completely transfer the contents. Note: The reconstituted Luciferase Sensor working solution is not stable. Protect from light.ProcedureRun Luciferase assay:Treat cells (or samples) with test compounds by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in desired compound buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.Incubate the cell plate in a 5% CO2 incubator at 37°C for desired period of time, typically 4 hours to overnight.Add 100 µL (96-well plate) or 25 µL (384-well plate) per well of Luciferase Sensor working solution.Incubate the plate at room temperature for 10 - 20 minutes. Keep from light.Monitor luminescence intensity with a luminometer.Establish standard Luciferase calibration curve: Note: Luciferase standard curve should be generated together with the above assay if the absolute amount of Luciferase in samples needs to be calculated.Make a series dilutions of Luciferase in PBS buffer with 0.1% BSA by including a sample without Luciferase (as a control) for measuring background luminescence. Note: Typically Luciferase concentrations from 1 pg/mL to 1 ng/mL are appropriate.Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of diluted Luciferase solution into an empty plate.Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Luciferase working solution.Incubate the reaction mixture at room temperature for 10 - 20 minutes, protected from light.Record the luminescence intensity with a standard luminometer.Generate the Luciferase standard curve.Data AnalysisThe reading (RLU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Luciferase samples. ImagesFigure 1. Luciferase dose response was measured with Amplite™ Luciferase Reporter Gene Assay Kit in a white 96-well plate with a NOVOstar plate reader (BMG Labtech). The kit can detect as low as 0.1pg/well luciferase with 20 minutes to 5 hours incubation without losing signal intensity. The integration time was 1 second. The half life is more than 4 hours.Figure 2. Reaction Kinetics of CHO-V2R-Luc cells using Amplite™ Luciferase Reporter Gene Assay Kit. CHO cells stably transfected with pCRE-luciferase gene and human Vasopressin receptor 2 (V2R) were plated into a 384-well white wall/clear bottom costar plate at 15,000 cells/well/25 µL. Cells then were treated with 100 nM of vasopressin in a 5% CO2 incubator at 37 °C for 4 hours. 25 µL of luciferase assay solution was added into the well. The kinetic data was taken every 30 minutes for up to 3 hours with a NOVOstar plate reader (BMG Labtech). The vasopressin induced luciferase signal is stable for more than 3 hours.