Exploring Applications of the Bioluminescent HiBiT Tag

Studying proteins in their natural context is one of the biggest challenges in biology. From tumor suppressors to growth factors, some of the most clinically-relevant proteins are also the hardest to study. One common strategy is protein overexpression — boosting levels so interactions aren't missed. But this often yields results that don't reflect true biology. Fluorescent fusions utilizing bulky tags can disrupt normal cell biology and make it hard to capture true protein behavior. Traditional epitope knock-ins maintain the native protein context, but they still depend on antibody-based detection or western blotting, methods that are low-throughput, semi-quantitative, and provide only snapshots rather than continuous views of protein activity. Together, these limitations create challenges for measurements needed to move discoveries from the lab bench into drug development. HiBiT technology helps close this gap!

Bridging discovery gaps with endogenous-level detection

HiBiT is a minimal 11-amino-acid fragment of the NanoBiT® complex, which, upon binding to its complementary partner LgBiT, reconstitutes a fully functional luciferase enzyme (Figure 1). Addition of substrate generates a proportionate luminescent signal, strong enough to detect from proteins expressed at their endogenous levels. HiBiT is compatible with both strong and weak promoters, and even modest endogenous pools generate high signal-to-background luminescence (Schwinn et al., 2018). The small size of HiBiT reduces interference with the native function of tagged proteins and facilitates precise CRISPR-based tagging, or low-levels of ectopic expression, thus avoiding artifacts common in overexpression models. Although HiBiT is often used with knock-in models, ectopic models provide researchers with a means to quickly iterate, work across cell models, and even introduce mutations. This vector-based use of HiBiT provides a means to quickly answer research questions before creating knock-ins. 

Figure 1: Schematic of HiBiT tagging technology.

The bioluminescence advantage

There are countless protein tags floating around the scientific world, so you might wonder what sets HiBiT apart. While the classic Green Fluorescent Protein (GFP) and other fluorescent proteins revolutionized live-cell protein detection, their bulk (~27 kDa) can perturb folding or trafficking and their detection can suffer from photobleaching and cellular autofluorescence (Tsien, 1998).

What sets HiBiT apart from these tags is its use of bioluminescence to reflect protein activity. Bioluminescence detection is a cornerstone of modern cell-based assays and enables ultra-sensitive detection of proteins without the challenges typically occurring with a fluorescence-based approach. These challenges can include background autofluorescence from the plates or media, which can obscure weak signals; photobleaching from the excitation light, causing the signal to gradually diminish over time; and, importantly, repeated excitation generating reactive oxygen species that can lead to phototoxicity, perturb biological processes, and compromise cell viability (Laissue et al., 2017). HiBiT overcomes these challenges and enables precise quantification with with nothing more than substrate addition and a simple add-mix-measure method.

NanoBiT® luciferase activity is compatible with both intra- and extracellular environments, making it effective for analyzing real-time kinetics, such as binding, degradation, trafficking (Hall et al., 2012). Antibody-based tools can also be combined with the HiBiT tag to provide complementary validation of protein expression and localization.

HiBiT-tagged targets in translational research

Researchers can incorporate HiBiT with a wide range of workflows, from classic overexpression to CRISPR knock-in, letting you track endogenous protein activity and measure responses to therapeutics in real time.

Let's take a closer look at several applications of this technology. In each section below, we cite the original paper but link to plasmids from the Promega Plasmid Collection that express similar constructs available at Addgene. Read the original paper for more detail on whether the authors incorporated HiBiT by CRISPR tagging at the endogenous locus or used other expression methods.

Cracking the code of 'undruggable' targets

Researchers can design drugs to bind to well-defined ligand-binding pockets or unique surface epitopes for many proteins, but some proteins do not have such epitopes. These classical 'undruggable' targets are often intrinsically disordered and structurally complex. The tumor suppressor TP53 (p53) is one example of these difficult-to-study genes. Its main function is to regulate DNA repair, senescence, and apoptosis. Work by Kamio et al. (2025) utilized HiBiT to explore TP53 biology. They first identified that in a set of patients with gastric cancer (GC), TP53 was one of the most frequently mutated genes.

Next, the authors explored how these variants of p53 shape chemosensitivity and mechanistic drug responses (Kamio et al., 2025). They designed HiBiT-tagged TP53 constructs, one with an R175H mutation to match a frequent mutation in early-onset GC and one with a mutation at R273H to match a mutation frequently found in late-onset GC. HiBiT-TP53 expression was detected by blotting with LgBiT, and the authors used a p53 response-element NanoLuc reporter to show that the mutants abolished transcriptional activity, even in the presence of endogenous wild-type protein. Together, the work links p53 mutations to treatment outcomes and provides a HiBiT-enabled framework for quantifying variant expression with a minimally disruptive tag.

Find HiBiT-TP53 Fusion Vector at Addgene!

Following extracellular RNA editors

Tracking factors that utilize exosomal transport is quite challenging. Work by Shibata et al. (2023) used HiBiT to measure the activity of ADAR1, which converts adenosine to inosine (A-to-I) within double-stranded RNA. They hypothesized that if ADAR1 were carried by extracellular vesicles (EVs), edited transcripts could endow neighboring cells with pro-oncogenic traits, facilitating metastatic spread. The authors used NanoBiT assays to quantify ADAR1-HiBiT in cell lysates, conditioned media, and purified EV fractions (Shibata et al., 2023). Luminescence signal was used to confirm efficient EV loading of ADAR1-HiBiT. When EVs from cells expressing ADAR1-HiBiT were added to LgBiT-expressing hepatocytes or co-cultured across very narrow filters, complementation in recipient cells proved that the enzyme was successfully transferred and internalized. Functional assays further showed that imported ADAR1 increased oncogenic editing of its most common target, AZIN1, linking EV-mediated protein spread to enhanced malignant potential. This work highlights HiBiT tagging as a sensitive reporter for tracking proteomic cargo flow via EVs.

Find ADAR-HiBiT Fusion Vector at Addgene!

Watching protein degradation in real time

Proteins involved in endoplasmic reticulum quality control navigate a large and interconnected system of dynamic and redundant roles. This makes attempts to purify or overexpress proteins inappropriate in discerning their true roles. Work conducted by Kamada et al. (2023) used HiBiT to characterize the proteasomal degradation of cystic fibrosis transmembrane conductance regulators (CFTR) (Kamada et al., 2023). CFTR is a membrane protein that essentially functions as an ion channel, mutations of which lead to cystic fibrosis, one of the most prevalent inherited diseases. The authors investigated the role of cytosolic ubiquitin ligases in the degradation of CFTR, specifically UBE3C. Degradation of CFTR was measured through decreases in HiBiT signal, which was slowed by knockdown of UBE3C or other components of the degradation pathway. HiBiT enabled the authors to observe degradation as well as endocytic trafficking in real time through extracellular-specific and full lytic measurements.

Find CFTR-HiBiT Fusion Vector at Addgene!

Real-time tracking of receptor internalization

Work conducted internally at Promega explored the use of HiBiT to track epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase essential for cell proliferation signaling. CRISPR knock-in HiBiT-tagged EGFR cells allowed researchers to track endogenous activity of EGFR, including dose-dependent response to EGF (Promega, 2025). The value of HiBiT in these experiments was the ability to track the internalization of EGFR in real time treatment with EGF. Due to the lack of membrane permeability of the LgBiT protein, it only binds HiBiT still present on the cell surface. EGF treatment resulted in rapid internalization and diminished luminescence signal, associated with endosomal localization of EGFR. This method offered kinetic, non-invasive monitoring of receptor dynamics at the cell surface.

Find HiBiT-EGFR and EGFR-HiBiT Fusion Vectors at Addgene!

Measuring messengers: growth factors

Growth factors present unique challenges for study. These secreted signaling proteins regulate vital cell and tissue processes, but their low abundance, complex regulation, and context-dependent activity can render traditional tagging approaches insufficient. Vascular endothelial growth factor A (VEGF-A) is one of these challenging proteins. VEGF-A mediates angiogenesis through its principal receptor tyrosine kinase VEGFR2 (also known as KDR), orchestrating blood-vessel formation. Precise, real-time pharmacology of this target has been hampered by transient interactions with co-receptors (like NRP1) and by endocytic trafficking of the receptor. Peach et al. (2021) utilized HiBiT to address these challenges, exploring the binding kinetics of the VEGF-A and the VEGFR2-NRP1 heteromer (Peach et al., 2021). They used HiBiT and LgBiT fusion proteins to track how specific modifications to the binding sites and various isoforms led to subtle changes in co-receptor dynamics, including NanoBRET assays to monitor the binding of fluorescently-labeled VEGF-A to the heteromeric receptor complex.

Find sHiBiT-KDR Fusion Vector at Addgene!

HiBiT advances detection of endogenous proteins

As researchers continue to improve more physiologically and endogenously relevant models, tools that increase confidence become more important. For many difficult-to-study protein targets, HiBiT provides an excellent solution compatible with high throughput processing and enables a wide range of detection capabilities. See a full list of detection solutions and HiBiT products offered by Promega.

The vectors linked above are ready to request from Addgene so you can combine the sensitivity of HiBiT with Addgene's quality control, support, and distribution capabilities. Give HiBiT a try and see your favorite proteins in a whole new light!

This post was written by Simon Moe, an Associate Product Marketing Manager at Promega. His work focuses on commercializing their cell and protein analysis products. You can find plasmids deposited by Promega scientists here.

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References and resources

References

Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R., Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P., & Wood, K. V. (2012). Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chemical Biology, 7(11), 1848–1857. https://doi.org/10.1021/cb3002478

Kamada, Y., Tateishi, H., Nakayamada, U., Hinata, D., Iwasaki, A., Zhu, J., Fukuda, R., & Okiyoneda, T. (2023). UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR. Cells, 12(23), 2741. https://doi.org/10.3390/cells12232741 

Kamio, T., Kono, Y., Hirosuna, K., Ozato, T., Yamamoto, H., Hirasawa, A., Ennishi, D., Tomida, S., Toyooka, S., & Otsuka, M. (2025). Genomic Differences and Distinct TP53 Mutation Site-Linked Chemosensitivity in Early- and Late-Onset Gastric Cancer. Cancer Medicine, 14(8). https://doi.org/10.1002/cam4.70793

Laissue, P. P., Alghamdi, R. A., Tomancak, P., Reynaud, E. G., & Shroff, H. (2017). Assessing phototoxicity in live fluorescence imaging. Nature Methods, 14(7), 657–661. https://doi.org/10.1038/nmeth.4344 

Peach, C. J., Kilpatrick, L. E., Woolard, J., & Hill, S. J. (2021). Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells. British Journal of Pharmacology, 178(12), 2393–2411. https://doi.org/10.1111/bph.15426

Promega Corporation (2025). Illuminating Transmembrane Proteins Using HiBiT CRISPR Cell Lines. Promega Notes. https://www.promega.com/resources/pubhub/2025/illuminating-transmembrane-proteins-using-hibit-crispr-cell-lines/

Shibata, C., Otsuka, M., Shimizu, T., Seimiya, T., Kishikawa, T., Aoki, T., & Fujishiro, M. (2023). Extracellular vesicle mediated RNA editing may underlie the heterogeneity and spread of hepatocellular carcinoma in human tissue and in vitro. Oncology Reports, 50(5). https://doi.org/10.3892/or.2023.8631

Tsien R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544. https://doi.org/10.1146/annurev.biochem.67.1.509 

More resources from Addgene

• Luciferase 101
• Luciferase Plasmid Collection
• Promega Plasmid Collection