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Amplite® Luciferase Reporter Gene Assay Kit *Maximized Luminescence*

Common 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.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells (samples) with test compounds (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  2. Add equal volume of Luciferase Sensor working solution
  3. Incubate at room temperature for 10 - 20 minutes
  4. Monitor luminescence intensity at 560 nm

Important notes
Thaw 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 SOLUTION

1. 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.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Run Luciferase assay:

  1. 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.

  2. Incubate the cell plate in a 5% CO2 incubator at 37°C for desired period of time, typically 4 hours to overnight.

  3. Add 100 µL (96-well plate) or 25 µL (384-well plate) per well of Luciferase Sensor working solution.

  4. Incubate the plate at room temperature for 10 - 20 minutes. Keep from light.

  5. 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.

  1. 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.

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of diluted Luciferase solution into an empty plate.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Luciferase working solution.

  4. Incubate the reaction mixture at room temperature for 10 - 20 minutes, protected from light.

  5. Record the luminescence intensity with a standard luminometer.

  6. Generate the Luciferase standard curve.

Citations

View all 11 citations: Citation Explorer
Use of Tox21 screening data to profile PFAS bioactivities on nuclear receptors, cellular stress pathways, and cytochrome p450 enzymes
Authors: Ooka, Masato and Sakamuru, Srilatha and Zhao, Jinghua and Qu, Yanyan and Fang, Yuhong and Tao, Dingyin and Huang, Ruili and Ferguson, Stephen and Reif, David and Simeonov, Anton and others,
Journal: Journal of Hazardous Materials (2024): 134642
SARS-CoV-2 omicron variants harbor spike protein mutations responsible for their attenuated fusogenic phenotype
Authors: Park, Seung Bum and Khan, Mohsin and Chiliveri, Sai Chaitanya and Hu, Xin and Irvin, Parker and Leek, Madeleine and Grieshaber, Ailis and Hu, Zongyi and Jang, Eun Sun and Bax, Ad and others,
Journal: Communications Biology (2023): 556
Discovery of small molecule agonists of the Relaxin Family Peptide Receptor 2
Authors: Esteban-Lopez, Maria and Wilson, Kenneth J and Myhr, Courtney and Kaftanovskaya, Elena M and Henderson, Mark J and Southall, Noel T and Xu, Xin and Wang, Amy and Hu, Xin and Barnaeva, Elena and others,
Journal: Communications biology (2022): 1--12
AtHSPR is involved in GA-and light intensity-mediated control of flowering time and seed set in Arabidopsis
Authors: Yang, Tao and Sun, Yan and Wang, Yongli and Zhou, Lina and Chen, Mengya and Bian, Zhiyuan and Lian, Yuke and Xuan, Lijuan and Yuan, Guoqiang and Wang, Xinyu and others,
Journal: Journal of experimental botany (2020): 3543--3559
AtHSPR is involved in GA-and light intensity-mediated control of flowering time and seed set in Arabidopsis
Authors: Yang, Tao and Sun, Yan and Wang, Yongli and Zhou, Lina and Chen, Mengya and Bian, Zhiyuan and Lian, Yuke and Xuan, Lijuan and Yuan, Guoqiang and Wang, Xinyu and others,
Journal: Journal of Experimental Botany (2020)

References

View all 52 references: Citation Explorer
Analysis of rhythmic gene expression in adult Drosophila using the firefly luciferase reporter gene
Authors: Stanewsky R., undefined
Journal: Methods Mol Biol (2007): 131
Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi
Authors: Blevins JS, Revel AT, Smith AH, Bachlani GN, Norgard MV.
Journal: Appl Environ Microbiol (2007): 1501
Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay
Authors: Li X, Shen F, Zhang Y, Zhu J, Huang L, Shi Q.
Journal: Eur J Pharm Biopharm (2007): 284
A GUS/luciferase fusion reporter for plant gene trapping and for assay of promoter activity with luciferin-dependent control of the reporter protein stability
Authors: Koo J, Kim Y, Kim J, Yeom M, Lee IC, Nam HG.
Journal: Plant Cell Physiol (2007): 1121
Reporter gene assay against lipophilic chemicals based on site-specific genomic recombination of a nuclear receptor gene, its response element, and a luciferase reporter gene within a stable HeLa cell line
Authors: Mori T, Saito F, Yoshino T, Takeyama H, Matsunaga T.
Journal: Biotechnol Bioeng. (2007)
Page updated on December 13, 2024

Ordering information

Price
Unit size
1 plate
10 plates
100 plates
Catalog Number
125181251912520
Quantity
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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC41105331

Platform

Luminescence microplate reader

Recommended plateSolid white

Components

Reaction Kinetics of CHO-V<sub>2</sub>R-Luc cells using Amplite® Luciferase Reporter Gene Assay Kit. CHO cells stably transfected with pCRE-luciferase gene and human Vasopressin receptor 2 (V<sub>2</sub>R) were plated into a 384-well white wall/clear bottom costar plate at 15,000 cells/well/25 &micro;L. Cells then were treated with 100 nM of vasopressin in a 5% CO<sub>2</sub> incubator at 37 &deg;C for 4 hours. 25 &micro;L 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.
Reaction Kinetics of CHO-V<sub>2</sub>R-Luc cells using Amplite® Luciferase Reporter Gene Assay Kit. CHO cells stably transfected with pCRE-luciferase gene and human Vasopressin receptor 2 (V<sub>2</sub>R) were plated into a 384-well white wall/clear bottom costar plate at 15,000 cells/well/25 &micro;L. Cells then were treated with 100 nM of vasopressin in a 5% CO<sub>2</sub> incubator at 37 &deg;C for 4 hours. 25 &micro;L 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.
Reaction Kinetics of CHO-V<sub>2</sub>R-Luc cells using Amplite® Luciferase Reporter Gene Assay Kit. CHO cells stably transfected with pCRE-luciferase gene and human Vasopressin receptor 2 (V<sub>2</sub>R) were plated into a 384-well white wall/clear bottom costar plate at 15,000 cells/well/25 &micro;L. Cells then were treated with 100 nM of vasopressin in a 5% CO<sub>2</sub> incubator at 37 &deg;C for 4 hours. 25 &micro;L 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.