A luciferase reporter assay is a common assay in molecular biology that uses the luciferase enzyme and a substrate (such as luciferin) to study gene regulation at the level of transcription.
Some important features of luciferase assays (Brasier & Ron, 1992):
- Extremely sensitive
- Compatible with internal control reporters
- The use of nonradioactive reagents
This article provides information about the luciferase assay, including the process and materials you will need to prepare before you perform the assays.
What is a Luciferase Reporter Assay?
A luciferase reporter assay is a test that investigates whether a protein can activate or repress the expression of a target gene using luciferase as a reporter protein (Carter & Shieh, 2015). The synthesis of the reporter protein and the addition of a substrate results in a chemical reaction with bioluminescence (or the emission of photons) as a by-product. This bioluminescence directly corresponds with the effect of the protein on expression of the target gene.
Examples of Bioluminescent Signal Measurements. When a protein of interest activates transcription, a luminometer reads a bright bioluminescent signal. However, if the protein represses transcription, the luminometer will detect no signal.
This type of assays can help you to test a functional connection between a protein and the amount of a gene product, but it is unable to test the direct interaction between a protein and a regulatory region of a target gene (Carter & Shieh, 2015).
Some applications of luciferase reporter assays are:
- Gene expression analysis
- Promoter structure analysis
- SNPs analysis
- Antiviral research and therapy
- Cytotoxicity assay
- Drug discovery (such as drugs targeting G-protein-coupled receptors)
How Does Luciferase Reporter Assay Work?
To perform a luciferase reporter assay, you will need a DNA plasmid to express the protein that you hypothesize could affect transcription.
You will also need to use another DNA plasmid with the regulatory element (or a target promoter region) fused with the DNA coding sequence for a luciferase enzyme. When activated, this system produces luciferase.
After cells are transfected with the luciferase reporter plasmid and
allowed to grow for few days, the next steps are to lyse the cells, add a
substrate to the cell lysate, and measure the luciferase activity based on the
amount of bioluminescent signal.
A Luciferase Reporter Assay. The luciferase assay is useful to study whether a protein of interest regulates a particular gene at the transcription level. By transfection, a DNA construct with the gene’s promoter and a coding region of the luciferase reporter gene enters the cells. Another DNA construct introduced into the cells consists of a coding region of the protein of interest. When this protein activates transcription, the cell will produce luciferase enzyme. After the addition of a lysis buffer and a substrate, a luminometer quantifies the luciferase activity.
If your protein activates the expression of the target gene, the amount of signal produced increases. However, if it blocks the gene expression, the cells produce less luciferase. As a result, these samples generate a lower signal than the positive control.
How to Choose a Cell Lysis Buffer for the Luciferase Assay
When choosing a buffer to lyse the cells for the luciferase assay, use ingredients that are compatible with the bioluminescent reagents. This means that the buffer must be able to protect the luciferase activity, so it won’t interfere with the activity of the bioluminescent reagent and the signal sensitivity.
For example, GoldBio luciferase lysis buffers retain sensitivity of the signal and work well with our Illumination™ luciferase assay kits. For Firefly luciferase reporter assays, use a luciferase lysis buffer, whereas for the Renilla luciferase reporter assays, choose a passive lysis buffer (PLB).
How to Make Luciferin Solution
Preparing a D-Luciferin Potassium or Sodium Stock solution
1. Prepare 15 mg/mL (100X) Luciferin stock solution in molecular biology grade H2O.
Note: For best results, the Luciferin stock solution should be used immediately, but it may be separated into small aliquots and stored at -80°C for up to 1 month.
2. The final concentration of Luciferin substrate in the Luciferase Luminescence Assay should be: 150 μg/ml (471μM for Luciferin-Potassium Salt, or 468μM for Luciferin-Sodium Salt).
Preparing a Coelenterazine Stock Solution
1. Add 1 mg coelenterazine to 1 ml of acidified methanol (add 50 µl of concentrated HCl to 10 ml methanol).
2. Aliquot and store at -80°C for up to 4 weeks.
3. For use, thaw tube and add 25.41 µl of coelenterazine stock solution to 40 ml DPBS (1.5µM final concentration).
How is Luciferase Activity Measured?
To measure luciferase activity of the cell lysates, you will need a multiwell plate or a tube containing cell lysates and a luminometer. This apparatus detects the photon emission produced from the luciferase reaction and the unit of measurement comes out as relative light units (RLU). Usually, for many luciferase assays, you won’t need to select particular wavelengths.
To increase the consistency of the results, it’s better to use a luminometer with an injector(s) for particular types of luciferase assays (such as dual luciferase reporter assays). The injector dispenses a substrate into the cell lysate and the reading of the wells starts immediately to minimize the possibility of diminishing signal (Smale, 2010).
D-Luciferin (Catalog ID: LUCK-100)
Coelenterazine (Catalog ID: CZ)
5X Passive Lysis Buffer compatible with Illumination™ Renilla Luciferase assay kit (Catalog ID: L-745)
5X Luciferase Lysis Buffer compatible with Firefly Luciferase assay kit (Catalog ID: L-740)
Luciferase Assay Kits:
Illumination™ Renilla Luciferase Enhanced Assay Kit (Catalog ID: I-925)
Illumination™ Firefly & Renilla Luciferase Enhanced Assay Kit (Catalog ID: I-920)
Illumination™ Firefly Luciferase Enhanced Assay Kit (Catalog ID: I-930)
Illumination™ Lyophilized Firefly Luciferase Enhanced Assay Kit (Catalog ID: I-935)
To learn more about luciferase assays, check our other articles:
Badr, C. E., & Tannous, B. A. (2011). Bioluminescence imaging: progress and applications. Trends in Biotechnology, 29(12), 624–633. https://doi.org/10.1016/j.tibtech.2011.06.010.
Brasier, A. R., & Ron, D. (1992, January 1).  Luciferase reporter gene assay in mammalian cells. ScienceDirect; Academic Press. https://www.sciencedirect.com/science/article/pii/007668799216036J.
Carter, M., & Shieh, J. (2015, January 1). Chapter 15 - Biochemical Assays and Intracellular Signaling (M. Carter & J. Shieh (eds.)). ScienceDirect; Academic Press. https://www.sciencedirect.com/science/article/pii/...
Cheng, Z., Garvin, D., Paguio, A., Stecha, P., Wood, K., & Fan, F. (2010). Luciferase Reporter Assay System for Deciphering GPCR Pathways. Current Chemical Genomics, 4, 84–91. https://doi.org/10.2174/1875397301004010084.
Gong, D.-W., Bi, S., Pratley, R. E., & Weintraub, B. D. (1996). Genomic Structure and Promoter Analysis of the Human obese Gene. Journal of Biological Chemistry, 271(8), 3971–3974. https://doi.org/10.1074/jbc.271.8.3971.
Hampf, M., & Gossen, M. (2006). A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Analytical Biochemistry, 356(1), 94–99. https://doi.org/10.1016/j.ab.2006.04.046.
He, M., Guo, H., Yang, X., Zhang, X., Zhou, L., Cheng, L., Zeng, H., Hu, F. B., Tanguay, R. M., & Wu, T. (2009). Functional SNPs in HSPA1A Gene Predict Risk of Coronary Heart Disease. PLoS ONE, 4(3), e4851. https://doi.org/10.1371/journal.pone.0004851.
Matta, H., Gopalakrishnan, R., Choi, S., Prakash, R., Natarajan, V., Prins, R., Gong, S., Chitnis, S. D., Kahn, M., Han, X., Chaudhary, V., Soni, A., Sernas, J., Khan, P., Wang, D., & Chaudhary, P. M. (2018). Development and characterization of a novel luciferase based cytotoxicity assay. Scientific Reports, 8. https://doi.org/10.1038/s41598-017-18606-1.
Min, J. S., Kim, G.-W., Kwon, S., & Jin, Y.-H. (2020). A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity. Journal of Clinical Medicine, 9(8), 2399. https://doi.org/10.3390/jcm9082399.
Nakajima, Y., Kimura, T., Sugata, K., Enomoto, T., Asakawa, A., Kubota, H., Ikeda, M., & Ohmiya, Y. (2005). Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. BioTechniques, 38(6), 891–894. https://doi.org/10.2144/05386st03.
Smale, S. T. (2010). Luciferase assay. Cold Spring Harbor Protocols, 2010(5), pdb.prot5421. https://doi.org/10.1101/pdb.prot5421.
Thorne, N., Inglese, J., & Auld, D. S. (2010). Illuminating Insights into Firefly Luciferase and Other Bioluminescent Reporters Used in Chemical Biology. Chemistry & Biology, 17(6), 646–657. https://doi.org/10.1016/j.chembiol.2010.05.012.