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Overcoming artifacts and capturing an accurate view of protein dynamics
Understanding how proteins behave in living systems is central to biology. Yet, for decades, researchers have relied on overexpression models or antibody-based detection methods that can distort the reality inside cells. Overexpression often drives protein expression above natural levels, leading to mislocalization, altered interactions or artificial function. Meanwhile, primary antibody performance varies widely across targets and experimental systems.
Endogenous tagging offers a more accurate view. By labeling proteins at their genomic locus, researchers can study proteins under native regulation, enabling direct monitoring of complex protein classes like transmembrane receptors and signaling proteins.
HiBiT knock-in cell lines improve on both of those methods by making it possible to measure and visualize proteins of interest exactly as expressed in the cell—with high sensitivity, quantitative precision, and no primary antibodies required.
Knock-in tagging has the upper hand
Knock-in vs. overexpression
When proteins are overexpressed from plasmids or viral vectors, results are often misrepresented by artifacts. Non-physiological expression levels may push proteins into receptors they don’t naturally occupy or alter their normal cellular interactions. Knock-in tagging avoids this issue by integrating a small reporter tag directly into the endogenous
locus using CRISPR/Cas9.
The HiBiT tag is 11 amino acids long and reconstitutes NanoBiT® luciferase upon binding its complementary LgBiT subunit, producing bright luminescence proportional to protein abundance. Compact tag size minimizes interference while the bright, stable signal enables detection at true endogenous levels.
Knock-in vs. antibody-based detection
Primary antibody-based detection remains essential but can introduce variability due to differences in antibody quality, specificity, and performance across assays. HiBiT tagging provides a simpler, more consistent alternative: the HiBiT tagging system delivers quantitative, reproducible measurements without primary antibodies or complex detection steps. Because luminescence detection is highly adaptable, the same tag supports multiple applications, from plate-based assays to live-cell imaging and kinetic studies. The HiBiT tag can also be detected using a highly specific, monoclonal antibody, ensuring compatibility with traditional epitope-tag methods like flow cytometry and immunoaffinity-based enrichment.
Explore protein stability, dynamics, and interactions
Endogenous tagging helps scientists spend less time troubleshooting and more time uncovering meaningful biological information. HiBiT Knock-In cell lines enable:
- Quantitative tracking of protein abundance across treatments or time
- Visualization of subcellular localization without overexpression
- Monitoring of stability, turnover, and degradation
- Exploration of endogenous protein complexes or post-translational modifications (PTMs) using immunoprecipitation mass spectrometry (IP-MS) approaches
Researchers at Promega used CRISPR/Cas9 to insert the HiBiT tag into an endogenous
Epidermal Growth Factor Receptor (EGFR) locus. The small luminescent tag allowed real-time monitoring of receptor internalization after ligand stimulation. Changes in luminescence directly reflected receptor activity and surface localization, providing rapid, quantitative data into a dynamic signaling process central to cell growth regulation. Additionally, researchers used IP-MS of EGFR-HiBiT to assess EGFR interactors and phosphorylation state. This same strategy can be extended to a wide range of proteins involved in signaling, degradation,
and receptor biology.
Easy paths to adoption
While CRISPR-based knock-in editing once seemed complex, recent advances have made endogenous tagging accessible for nearly any lab. Researchers can choose from three methods:
- Ready-to-use HiBiT knock-in cell lines for rapid data generation and validation.
- Custom tagging services and kits for specific or unique genes or models.
- Build-your-own cell lines for full experimental control: synthesize donor DNA and obtain the rights to incorporate the HiBiT tag using a straightforward protocol.
These flexible solutions meet researchers where they are: whether exploring a single
target or scaling up for high-throughput detection and screening.
Conclusion
Endogenous tagging combines CRISPR/Cas9 precision with bioluminescent sensitivity for accurate, reproducible protein characterization, avoiding the limits of primary antibody detection while supporting downstream antibody use. This approach delivers a clearer view of protein biology that continues to drive progress in translational research and drug discovery.
Learn more about CRISPR Knock-In Tagging at: www.promega.com/CRISPR-HiBiT.
The post The Case for Endogenous Tagging appeared first on GEN – Genetic Engineering and Biotechnology News.
