Simplicity, Sensitivity, and Speed in One Tag
From drug screening to pathway validation, the ability to monitor endogenous protein expression quickly and quantitatively is a cornerstone of modern cell biology. Yet many researchers still rely on outdated tagging methods that are semi-quantitative at best—or not compatible with live-cell workflows.
HiBiT is changing that. And if you're still using legacy tags like FLAG or HA—or building GFP fusions that disrupt protein function—this post is for you.
What is HiBiT, and Why Should You Care?
HiBiT is an 11-amino-acid peptide tag that, when combined with its complementary partner LgBiT (provided in Promega's detection reagents), reconstitutes an active luciferase enzyme. The result? A bright, quantitative luminescent signal directly proportional to the amount of tagged protein in the sample.
It’s that simple—and that powerful.
How HiBiT Outperforms Conventional Tags
With HiBiT, you eliminate the headaches of antibody validation, signal variability, and overexpression artifacts. Whether you're working with engineered cell lines or transgenes, HiBiT gives you functional fidelity—you tag your protein without compromising its behavior.
Real-World Example: Using HiBiT to Uncover the CK2–CARD9 Axis in Innate Immunity
In a recent study out of Bristol Myers Squibb, researchers sought to understand the upstream regulation of CARD9—a critical adaptor protein in antifungal innate immune signaling. While CARD9’s role in activating inflammatory pathways was well established, the molecular mechanisms controlling its stability and degradation remained poorly defined.
To solve this, the team used EditCo to generate a CRISPR knock-in THP-1 cell line with endogenous CARD9 tagged using HiBiT. This enabled real-time, quantitative measurement of native CARD9 protein levels via luminescence—without the need for antibodies or overexpression artifacts.
With this platform in place, the team performed a targeted CRISPR knockout screen of individual casein kinase 2 (CK2) isoforms and subunits. The results were striking: loss of CK2α (CSNK2A1) and CK2α′ (CSNK2A2) significantly stabilized CARD9 protein levels, while other CK2 components had minimal or no effect. This revealed, for the first time, that phosphorylation by CK2α/α′ drives CARD9 degradation—establishing a direct link between kinase activity and CARD9 protein turnover.
HiBiT’s high sensitivity and compatibility with arrayed genetic perturbation enabled rapid, mechanistic profiling of immune signaling pathways that would have been far more laborious using traditional Western blot or antibody-based approaches.
EditCo Highlight: Endogenous HiBiT tagging revealed CK2α/α′ as critical regulators of CARD9 stability, unlocking new insights into immune signaling and enabling translational exploration of this pathway for autoimmune disease.
Use Cases Where HiBiT Shines
Whether you're working in drug development or basic discovery, HiBiT opens up new possibilities:
- Target Engagement Assays
Quantify drug-induced degradation or stabilization of your target protein in live cells.
- Mechanism-of-Action Studies
Monitor how your perturbation affects protein expression, turnover, or cellular localization.
- Pathway Mapping
Track protein changes in real time across signaling cascades—no blotting required.
- Gene Regulation Screens
Quantify transcriptional and post-translational effects with luciferase-like sensitivity.
- Cell Therapy Research
Monitor persistence or differentiation markers with minimal cellular perturbation.
Why Outsource Your HiBiT Knock-ins?
HiBiT sounds great—until you realize your team doesn’t have the capacity to make CRISPR knock-in lines, especially if you're working with hard-to-edit cell types or tight timelines.
That’s where EditCo comes in.
Through our partnership with Promega, EditCo now offers custom HiBiT-tagged cell lines—with:
- CRISPR/RNP precision (no plasmids)
- NGS-validated integration with optional functionality testing
- Licensing coverage handled on your behalf
- Short delivery timelines aligned with drug discovery sprints.
Whether you’re tagging an endogenous gene or introducing a tagged transgene, we handle the heavy lifting so you can stay focused on the science.
Frequently Asked Questions
Q: We already use FLAG or HA. Why switch?
HiBiT offers true quantitation, live-cell compatibility, and removes the need for validated antibodies. It’s not just a convenience—it’s a fundamentally more scalable, reproducible system.
Q: Can I use HiBiT in primary or iPS cells?
Yes. We’ve successfully integrated HiBiT into primary fibroblasts, neural progenitors, iPSCs, and other challenging models. Ask us about your specific cell type.
Q: What about detection? Is the assay complicated?
Detection is easy. Promega’s detection kits supply the LgBiT protein and substrate. Add reagent, incubate for 10 minutes, and read luminescence. That’s it.
Q: We already did a HiBiT knock-in before—do we need to worry about licensing now?
With our Promega licensing agreement, EditCo now covers your use of HiBiT in our custom cell lines—removing the burden of compliance and making future projects seamless.
Ready for custom HiBiT knock-ins?
Request a quote today!
