Why Endogenous Tagging Matters
Most tagging strategies rely on plasmid-based expression or bulky protein fusions. While easy to deploy, these systems introduce artifacts, mislocalization, and variability.
Figure Legend: Cells are not empty spaces, they are molecular cities. What you’re seeing here are three snapshots of a eukaryotic cell: protein sorting in the Golgi, vesicle transport, and nuclear pore transport. Each illustration is built from high-resolution molecular imaging and structural data, and every shape you see represents real proteins, drawn to scale. Source: Eukaryotic Cell PanoramaDavid S. Goodsell, Biochemistry and Molecular Biology Education Vol. 39, No. 2 pp. 91-101, 2011
Most of us were first taught biology with simplified analogies: a cell as an egg, organelles as little compartments, proteins floating freely like beads in a jar. But this isn’t how biology actually works.
Inside a living cell, there is no unused space.
Proteins are packed shoulder-to-shoulder, interacting, binding, and competing in a tightly regulated choreography. The Golgi is not just a stack of membranes, it’s a dense sorting hub where enzymes, scaffolds, and trafficking proteins work together with exquisite precision. Vesicle transport isn’t just “bubbles moving cargo”, it’s a motor-driven logistics network that relies on coat proteins, adaptors, receptors, and cytoskeletal highways. And the nuclear pore is not a hole, it is one of the largest protein assemblies in the cell, made of hundreds of subunits that control every molecule entering or exiting the nucleus.
This level of complexity matters. In an environment this crowded and dynamic, biology is highly sensitive to perturbation. Overexpression models, bulky tags, or artificial constructs can easily distort natural interactions. A small change to a protein’s size, localization, or stability can ripple across entire pathways.
That’s why precision engineering is foundational to our approach.
By integrating small functional tags directly at the endogenous locus, without disrupting expression, folding, or trafficking, we create models that behave the way real proteins behave inside real cells. In a system this dense and coordinated, precision isn’t optional. It’s the difference between getting noisy artifacts… and uncovering biology as it actually operates.
Precision in, precision out.
That’s what lets our tagging strategy deliver real insights from this complexity.
EditCo delivers small, functional tags precisely inserted at endogenous loci, unlocking:
- True biological behavior - no artificial overexpression
- Reliable reproducibility - sequence-verified, stable integration
- Cleaner workflows - compatible with fluorescence, luminescence, and antibody systems
Endogenous tagging provides a unified, reproducible way to quantify, visualize, purify, and study protein turnover, without compromising biology.
The Four Pillars of Protein Tagging
A flexible tagging platform built for every assay.
Different biological questions require different functional readouts, and no single tag can answer all of them. That’s why EditCo’s tagging platform is intentionally built around four foundational pillars: Visualization, Quantification, Purification, and Degradation.
Each pillar represents a distinct way to interrogate proteins inside their native environment, and each relies on tags designed to preserve biological fidelity while revealing specific mechanistic insights.
1. Visualization
See proteins in the context of their native environment.
In a cell as crowded and structured as the Golgi, ER, cytoskeleton, or nucleus, localization is more than just “where a protein is.” It’s a proxy for function, interaction partners, trafficking state, and signaling logic. Disrupting localization, even subtly, can fundamentally rewrite a protein’s role.
Figure Legend: This image illustrates how fluorescent and covalent protein tags enable high-resolution visualization of protein localization within the crowded and highly structured environment of the cell. Rather than existing in isolation, proteins operate within dense networks of membranes, scaffolds, and interaction partners, where spatial positioning directly influences function. Endogenous tagging strategies preserve native expression and trafficking, allowing researchers to observe proteins exactly where they operate, revealing localization patterns, dynamic movement, and spatial relationships that are often distorted by overexpression-based systems.
Endogenous luminescent, fluorescent, and covalent tags allow you to visualize proteins exactly where they operate, without the mislocalization, aggregation, or artifacts often introduced by overexpression or oversized fluorescent fusions.
With precise HiBiT, GFP, mCherry or your florophore of preference knock-ins, you can track:
- How proteins move through organelles
- How trafficking adapts to perturbations
- How spatial relationships influence pathway logic
- How dynamic environments reshape protein functionality
Visualization isn’t just about seeing, it’s about understanding structure, context, and temporal behavior inside the molecular city of the cell.
2. Quantification
Turn cellular behavior into measurable, dynamic data.
In biology, abundance is not static, proteins are synthesized, modified, transported, activated, and degraded continuously. To understand mechanism, you need a readout that reflects real-time kinetics, not endpoint snapshots.
Figure Legend: Cells constantly tune protein abundance in response to environmental cues, stabilizing some proteins, degrading others, and these changes often occur within minutes. The two graphs shown here illustrate how endogenous luminescent tagging with HiBiT enables highly sensitive, quantitative measurement of these dynamics. Left: HIF1A is normally kept at very low levels through rapid hydroxylation and ubiquitin-mediated degradation. When cells encounter hypoxia or hypoxia-mimetic compounds such as 1,10-phenanthroline, HIF1A stabilizes and accumulates quickly. Right: BRD4 is a well-studied target for PROTAC and TPD approaches. When exposed to the degrader dBET1, BRD4 is rapidly ubiquitinated and destroyed. Source: https://www.promega.com/resources/pubhub/2017/quantifying-protein-abundance-at-endogenous-levels/
Endogenous luminescent tags like HiBiT and NanoLuc transform protein abundance into precise, quantitative signal. Because the tag sits directly at the native locus, every change in signal corresponds to a true biological event, not variations in transfection efficiency or expression noise.
This enables:
- Live-cell kinetic measurements with second-level temporal resolution
- Direct monitoring of protein stabilization or destabilization
- Real-time response profiling across perturbations
- High-throughput screening with quantitative confidence
Quantification is the foundation of mechanism-of-action, dose-response, and temporal biology. In a system where everything is in motion, dynamic measurement is essential.
3. Purification
Pull down the proteins that matter, without rewriting their biology.
When proteins exist in dense molecular ecosystems, interactions and stoichiometries are exquisitely sensitive. Overexpressing tagged proteins can overwhelm complexes, disrupt binding equilibria, or artificially create new interactions that never occur in vivo.
Endogenous epitope tags, such as HiBiT, FLAG, HA, and Myc, allow researchers to isolate proteins exactly as they exist in living cells: native partners, natural post-translational modifications, real stoichiometries, and physiological abundance.
Figure Legend: Endogenous HiBiT tagging enables high-affinity immunoprecipitation of BRD2, PARP1, and BTK at native expression levels. Anti-HiBiT Magne® Beads efficiently deplete tagged proteins from the lysate and enrich them in the eluate, preserving natural stoichiometries and interactions for IP, co-IP, Western blotting, and proteomics. Source: https://www.promega.com/products/protein-purification/protein-purification-kits/anti-hibit-magnetic-beads-and-elution-peptide/?tabset0=0
With endogenous purification tags, you can:
- Perform IP and Western blotting without overexpression artifacts
- Capture real interaction partners and avoid non-physiologic complexes
- Study proteoforms and modification states as they naturally occur
- Generate clean biochemical datasets grounded in native biology
Purification isn’t just for confirmation, it’s a window into true molecular architecture.
4. Degradation
Control protein stability with precision-engineered degradation tags
Protein degradation systems like dTAG and HaloPROTAC rely on exquisitely tuned molecular interactions. To work effectively, the degradation handle must be small, accessible, and correctly folded, at the protein’s native expression level.
Overexpression-based dTAG constructs or large fusion proteins often misfold, mislocalize, or compete with endogenous protein, leading to misleading degradation profiles.
Figure Legend: Schematic overview of HaloPROTAC function. Bottom: This figure illustrates how endogenous HaloTag knock-ins enable precise, conditional protein degradation using HaloPROTAC3. HaloPROTAC3 is a bifunctional degrader that binds HaloTag-fused proteins via a chloroalkane moiety while simultaneously recruiting the endogenous VHL E3 ligase complex, triggering ubiquitination and proteasomal degradation. Unlike genetic knockouts or permanent protein mutations, HaloPROTAC-mediated degradation provides temporal control over protein loss, allowing researchers to study phenotypes that depend on the timing, magnitude, and reversibility of degradation. When HaloTag is integrated directly at the endogenous locus using CRISPR/Cas9, degrader engagement occurs at physiological expression levels, avoiding misfolding, mislocalization, and competitive artifacts associated with overexpression systems. Kinetic measurements show rapid, ligand-induced loss of target protein, followed by sustained degradation over time. These dynamics highlight how endogenous HaloTag models faithfully capture degradation behavior in live cells, enabling mechanistic studies of protein stability, turnover, and functional rescue in relevant biological contexts. Together, these data demonstrate how precision-engineered HaloTag knock-ins provide a biologically accurate platform for targeted protein degradation studies, supporting translational research in drug discovery and chemical biology. Source: https://www.promega.com/applications/small-molecule-drug-discovery/protein-degradation-drug-discovery/degradation-phenotype/
By integrating HaloTag directly at the endogenous locus, EditCo enables true conditional degradation that reflects real biology, not an engineered artifact.
Endogenous HaloTag allows researchers to:
- Induce rapid, selective degradation with high temporal precision
- Compare KO vs conditional degradation in the same genetic background
- Study stability, rescue, and turnover mechanisms in native context
- Evaluate degrader potency and selectivity without expression noise
Degradation is one of the most powerful ways to probe causality in biological systems — and endogenous HaloTag models bring that power firmly into the realm of translational relevance.
| Pillar | Biological Question | Tag Modalities | Representative Examples | Why Endogenous Matters |
| Visualization | Where is the protein, and how does it move within the cell? | Fluorescent tags, covalent labeling tags | HiBiT, HaloTag, GFP, mCherry | Preserves native folding, localization, and trafficking in crowded cellular environments |
| Quantification | How much protein is present, and how does it change over time? | Luminescent or enzymatic reporters | HiBiT, NanoLuc | Signal reflects true biological change, not expression noise or transfection efficiency |
| Purification | What does the protein interact with, and in what stoichiometry? | Epitope tags, affinity tags, enzyme tags | HiBiT, FLAG, HA, Myc, ALFA | Captures native complexes, PTMs, and physiologic interaction partners |
| Degradation | When and how is the protein removed from the system? | Ligand-inducible handles | HaloTag-based dTAG / HaloPROTAC systems | Enables precise temporal control at endogenous expression levels |
Case Study Highlight
Visualizing the Role of PARP7 in Innate Immunity
Endogenous HiBiT knock-ins enabled real-time visualization and quantification of PARP7 expression dynamics in immune signaling pathways. By preserving native regulation, this approach revealed how PARP7 abundance and localization change during innate immune activation, insights that are often obscured by overexpression-based models.
Full case study coming soon! Stay tuned for detailed results and methodology.
