Bringing Luciferase from Nature to the Benchtop
Research and Product Spotlight
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.
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.
The 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.
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-Luciferase
pGL 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 System
Bright-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 Assay
CellTiter-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™
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
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
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
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