Celebrating 30 Years of Innovation and Discovery using Bioluminescent Technology
Bringing Luciferase from Nature to the Benchtop
Join industry leaders and Promega scientists in this webinar series to learn about recent advancements in important research topics such as Targeted Protein Degradation, GPCR and Kinase Biology, and CRISPR/Cas9 Knock-in Tagging.
Research and Product Spotlight
NanoBiT® Technology
NanoLuc® Luciferase has been engineered into a complementation reporter system termed “NanoLuc Binary Technology" or NanoBiT. The NanoBiT® system consists of an optimized large subunit from NanoLuc, LgBiT, along with small 11-amino-acid subunits that have variable affinity for the LgBiT subunit. Structural complementation of both subunits reconstitutes a bright luciferase enzyme.
NanoBiT® technology creates new possibilities for using bioluminescence to study protein dynamics. Using the low affinity small subunit, SmBiT, sensitive live-cell protein:protein interaction assays can be created by fusing LgBiT and SmBiT to target proteins of interest. In contrast the high affinity small subunit, HiBiT, self-associates with the LgBiT peptide, making HiBiT an easily detected and highly sensitive protein tag. The HiBiT protein tagging system can be used for both intracellular and extracellular protein detection, and when combined with CRISPR-based tagging, allows creation of knock-in reporter models that accurately reflect endogenous protein biology. Most recently NanoBiT has been further adapted into Lumit™ Technology for detection of specific antigens or antibodies, bringing the power of bioluminescence to immunoassay applications.
NanoBRET™ Technology
The small size and bright light output from NanoLuc are ideal characteristics for use as a protein tag. Together with a narrow, blue-shifted emission spectrum, these qualities make NanoLuc an ideal energy donor in Bioluminescence Resonance Energy Transfer (BRET) applications. Combined with red-shifted energy-accepting fluorophores, the NanoBRET™ system has optimal spectral overlap, increased signal and lower background compared to conventional BRET assays.
These fluorophores can be added to molecules such as protein ligands to create NanoBRET™ Target Engagement assays for measuring small molecule binding to target proteins or configured as HaloTag® ligands for monitoring protein:protein interactions in live cells using Nanoluc® and HaloTag® fusion proteins. Live-cell substrate options allow for a stable NanoLuc donor signal that can last for hours to days, enabling new opportunities for understanding protein dynamics within the cellular environment.
Watch how researchers from Keele University have used a NanoBiT® PPI assay to screen for novel autophagy inhibitors.
Watch how Aurelia Bioscience is using NanoBRET™ Target Engagement to screen Kinase-binding compounds in live cells.
NanoLuc® Luciferase
The introduction of NanoLuc® (Nluc) luciferase further expands bioluminescent reporter assay options and applications. The 19.1kDa NanoLuc® enzyme was evolved from the small subunit of the native luciferase from the deep sea shrimp, Oplophorus gracilirostris. The substrate was independently evolved into the optimized substrate furimazine, which together with the NanoLuc® enzyme create a reporter system that is about 100-fold brighter than either firefly or Renilla luciferases. In addition to the small size and high intensity, glow-type luminescence; NanoLuc is ATP-independent, has high thermal stability and activity over a broad pH range and has no post-translational modifications or disulfide bonds.
NanoLuc has been applied to traditional reporter applications as a single reporter or in a dual-assay format with firefly luciferase. However, due to its unique properties, researchers have found many non-traditional applications for this small, bright reporter. For example, its size makes it well suited to integration into small viral genomes where it has been used in the creation of a wide variety of sensitive reporter viruses. These have been used in both plate-based screening and in vivo imaging applications, including important research into SARS-CoV-2 biology and potential therapies.
In addition to using the firefly luciferase reaction to measure the amount of luciferase or ATP in a sample, it is also possible to measure changes in substrate (luciferin) concentration. By coupling the reaction with pro-luciferin substrates containing protecting groups that can be reacted upon by different enzyme classes, sensitive add-and-read assays have been created to measure the activity of various proteases and metabolizing enzymes, where the amount of light produced is proportional to enzyme activity. The Caspase-Glo® 3/7 Assay System was an early example of this approach, allowing for a simple, sensitive method to detect cells undergoing apoptosis. The ability to easily measure apoptotic cell death has allowed oncology researchers to efficiently screen for and characterize potential therapeutic approaches that can lead to the removal of cancer cells. Recently, this approach has been further adapted to enable the measurement of apoptosis in 3D culture systems, allowing scientists to study induction of apoptosis in model systems that more accurately mimic tumor cell growth.
Learn more about measuring apoptosis in 3D culture systems ›
Read how Nobel laureate Gregg L. Semenza used dual-reporter assays to advance our understanding of hypoxic gene regulation.
Read About Semenza's WorkThe Luciferase Assay System (LAR) was the first luciferase detection reagent introduced by Promega in 1991 along with the firefly luciferase, luc, reporter gene. The LAR assay still provides one of the brightest firefly luciferase detection solutions with flash signal kinetics that require injector delivery. Over the last 30 years the LAR reagent has been used in thousands of research projects to advance our understanding of topics ranging from regulation of gene expression to functional analysis of genetic polymorphisms. Today researchers are finding reagents like LAR to be critical tools in our efforts to identify vaccines and treatments for SARS-CoV-2 infection.
Blast from the Past
Read our first review and announcement about luciferase reporters in its original formfrom 1990.
Firefly Luciferase Sheds Light on Development of New Malaria Treatments
Read About Harnessing the Power of Firefly Luciferase
See how Dr. Paul Horrocks uses a firefly luciferase-based system to understand the dynamics of drug action in the development of new malaria treatments.
First generation luciferase detection assays have very bright but short-lived signal and require lysate generation prior to addition of assay reagents. Although extremely powerful, the short signal duration and upstream processing requirements present challenges to researchers using bioluminescent reporters in high-throughput analysis. The second generation “Glow” reagents such as the Bright-Glo™ and Steady-Glo® Assay Systems overcame these limitations by extending the kinetics of the firefly luciferase reaction (30 minute and 5-hour respective half-lives) while also providing a single-addition reagent that is added directly to cells in culture to lyse the cell and provide all of the assay components. These “add-mix-measure” assays significantly simplified sample processing and brought compatibility to microplate workflows allowing researchers to batch process 10s to 100s of plates at a time, expanding the use of luciferase reporter assays to high-throughput screening applications.
Learn more about the differences between Flash and Glow assays ›
ATP is well recognized as an indicator of metabolically active cells, making the measurement of total ATP levels an important tool in determining the impact of compound treatment on cell viability. The CellTiter-Glo® Luminescent Cell Viability Assay uses the cell as source of the ATP in the luciferase reaction, producing a bioluminescent signal that is proportional to the number of viable cells in a well. Similar to other “Glow”-type luciferase assays, the luminescent signal has an extended half-life ( > 5hr), and the reagent can be added directly to cells in culture making it ideal for automated high-throughput cell proliferation and cytotoxicity assays. The assay has been further optimized to reduce preparation time and simplify repeat use in the CellTiter-Glo® 2.0 assay and has also been adapted to measuring cellular ATP in 3D model systems for determining cell viability in these more complex physiological models.
See how Dr. Samantha Llewellyn uses CellTiter-Glo® 3D to assess toxicity of in physiologically relevant 3D liver models.
A Glo-ing History of Innovation and Discovery
Looking back at 30 years of bioluminescent discovery, innovation, and ground breaking research.
Luciferase Assay System
The first luciferase detection reagent introduced by Promega providing the beginning of sensitive, non-radioactive reporter gene assays. The LAR assay still provides one of the brightest firefly luciferase detection solutions with flash signal kinetics that require injector delivery. LAR along with the firefly luciferase (luc) reporter gene, provided some of the first tools that allowed researchers to begin mapping regulators of gene expression.
Read about Bioluminescent Reporter Genes
Dual-Luciferase® Reporter Assay System (DLR)
DLR was the first reagent that allowed for sequential detection of a second reporter in a single sample. It provided a key advancement in improving reporter assay reliability by allowing for internal normalization of luciferase activity.
See how Scientists are Applying Dual-LuciferasepGL Luciferase Reporter Vector Series
The pGL3 family of reporter vectors featured a modified firefly luciferase gene, luc+. This early example of engineering a reporter for performance improvements was later taken further for the pGL4 and luc2 reporters with even great improvements possible through bioinformatics and synthesis.
pGL4 Luciferase Reporter Vectors Tool
ENLITEN®/Ultra-Glo™ Recombinant Luciferase
Promega offered a recombinant firefly luciferase (ENLITEN) early on, but through an early example of directed evolution, a thermostable luciferase was engineered, called Ultra-Glo. The development of Ultra-Glo was key to making one-step, add-and-read assays with a variety of assay and storage conditions.
ENLITEN® ATP Assay SystemBright-Glo™ (1999), Steady-Glo® (1998), Dual-Glo® (2001) Luciferase Assay Systems
Through the development of new ways to alter the signal kinetics of the firefly luciferase assay, Bright-Glo, Steady-Glo, and Dual-Glo allowed use of microtiter plates for assays. The add-and-read format simplified sample processing and allowed use of reporter gene assays in very high-throughput applications.
Find the Best Reporter AssayCellTiter-Glo® Cell Viability Assay
With the development of Ultra-Glo luciferase, it was now possible to make an add-and-read ATP detection assay. ATP has been found to be a key indicator of cell health, making CellTiter-Glo a powerful assay for assessing cell viability especially in higher-throughput applications. The assay principle also lead to other platforms that measure ATP, notably Kinase-Glo (2004) and ADP-Glo (2009) enzyme assays used to study ATPases such as kinases.
See How CellTiter-Glo is being used in COVID-19 Research
Caspase-Glo® 3/7 Assay
In addition to measuring the amount of luciferase or ATP in a sample using the firefly luciferase reaction, it is possible to measure changes in substrate (luciferin) concentration. By coupling luciferin with protecting groups that can be reacted upon by different enzyme classes, sensitive, add-and-read assays are possible for these enzymes. Examples include Caspases and other proteases, and Cytochrome P450s.
Using Caspase-Glo® Assay in Cancer Research Explore Caspase-Glo®
ONE-Glo™ Luciferase Assay System
With further understanding of the firefly luciferase reaction chemistry and a team of biologists and chemists at Promega, an improved luciferin was created to be better suited for use in typical reporter gene assay applications. This new substrate, fluoroluciferin, is an early example of novel substrate development.
Pseudotyped Viral Particles and Luciferase Discover ONE-Glo™
NanoLuc® Luciferase
Experience in directed evolution and development of novel substrates came together to design a new luciferase reporter. system Adapted from a shrimp luciferase, the new NanoLuc luciferase was developed to be a small (19kDa) monomer with a unique substrate that offers approximately 100x great sensitivity than the already highly sensitive firefly or Renilla luciferase systems. This novel reporter would be used in many applications and serve as the basis for further technological development.
Researchers use NanoLuc Luciferase to Create Models of Cardiovascular Disease
NanoBRET™ Technology
The efforts to develop NanoLuc luciferase resulted in a versatile platform for further developments. The small size and very bright light output from NanoLuc was recognized to offer ideal characteristics as a protein tag. These traits also serve well as a donor for Bioluminescence Resonance Energy Transfer (BRET). A thorough study of a variety of energy-accepting fluorophores found options in the red spectrum that helped eliminate some of the challenges associated with BRET measurements. These fluorophores could be added to molecules such as protein ligands to measure engagement of a target protein or configured as HaloTag ligands to allow detection of protein:protein interactions in live cells.
Using NanoBRET to Better Understand the Kinome
NanoBiT® Technology
With the successful design of NanoLuc, scientists at Promega then endeavored to configure this reporter into a multi-subunit system. The resulting system, termed "NanoLuc Binary Technology" or NanoBiT, is a two part system consisting of an 11 amino acid small tag and a larger, further refined subunit of NanoLuc, LgBiT. Structurally complementation of both parts reconstitutes a bright luciferase enzyme. Affinity of the these subunits can be low as with SmBiT peptide, allowing for creation of protein interaction assays. Or it can be high, as with HiBiT, allowing for self assembly. HiBiT serves as an easily detected, highly sensitive protein tag that, when used with CRIPSR-based tagging, allows for the creation of endogenous reporter models.
Adapting NanoBiT to a Biochemical Assay Format
Lumit™ Technology
Following on the development of NanoBiT technology, it was recognized that this system could be used to detect a wide variety of analytes through conjugation of components of immunoassays. The resulting platform, now called "Lumit", offers simplified immunoassays with high sensitivity.
Explore Lumit™ Technology
Bioluminescence Applications Guide
Download select chapters from our Bioluminescence Applications Guide to learn more about Genetic Reporters, NanoLuc® Luciferase, and Plate Reader options. Request your full copy of the guide.
Present, Meet the Past
The Turner TD-20/20 was one of the first luminometers to offer dual-injectors. This made it a useful instrument for performing the Dual-Luciferase Assay.
If you think this is cool, check out the tool that aided Promega's early bioluminescent protein research.
Helping Researchers Analyze Luminescence and Fluorescence Data
