PROTAC Degradation Activity Detection Services

What Is PROTAC Degradation Activity Detection?

PROTAC (proteolysis-targeting chimera) degradation activity detection is a specialized cell-based assay that quantifies the ability of a bifunctional molecule to recruit an E3 ubiquitin ligase to a target protein, triggering its ubiquitination and subsequent proteasomal degradation. Unlike traditional inhibition assays that measure loss of enzymatic function, degradation assays directly measure the disappearance of the target protein from cells. This approach is essential for determining the degradation efficiency (DC50 and Dmax) and kinetic profile of candidate PROTACs, enabling structure-activity relationship (SAR) optimization and lead selection.

Our service employs multiple detection platforms—including split luciferase (HiBiT), time-resolved FRET (TR-FRET), and Western blot—to accommodate different target protein characteristics and project requirements. We work with a range of mammalian cell lines, including HEK293, HeLa, or target-expressing lines, and can engineer HiBiT or GFP tags endogenously via CRISPR to enable high-throughput screening. The assay can be performed in multiwell plates (96- to 384-well formats) with multiple PROTAC concentrations and time points to generate robust degradation curves.

Beyond primary degradation measurement, we also offer counter-screening for ternary complex formation using TR-FRET binding assays, as well as cell viability normalization to ensure that observed protein loss is not due to cytotoxicity. This comprehensive characterization is critical for advancing PROTAC molecules from hit identification to preclinical development.

Our PROTAC Degradation Activity Detection Platforms

HiBiT Luminescence Assay

Endogenous HiBiT tagging via CRISPR knock-in allows real-time, split-luciferase-based detection of residual target protein after PROTAC treatment. This high-throughput platform provides sensitive, quantitative readouts in 96-/384-well formats with minimal cell handling.

TR-FRET / HTRF Degradation Assay

Homogeneous time-resolved FRET using target-specific antibodies or tagged proteins enables medium- to high-throughput measurement of target degradation in live or lysed cells. Suitable for both endogenous and overexpressed targets as scoped per project.

Western Blot / Simple Western Validation

For orthogonal confirmation or when antibody-based detection is preferred, we perform traditional Western blot or automated capillary-based Simple Western (Jess) assays to visualize full-length target reduction and confirm degradation specificity.

Ternary Complex Binding Counter-Screen

A TR-FRET-based binding assay that measures the formation of a ternary complex among PROTAC, target protein, and E3 ligase. This orthogonal assay helps confirm the degradation mechanism and distinguishes true degraders from nonspecific binders.

How It Works: PROTAC Degradation Activity Detection Workflow

Cell Culture and Target Tagging We culture the appropriate mammalian cell line—either with endogenous target expression or engineered to express a HiBiT- or GFP-tagged version of the target protein. For stable cell lines, we generate clonal populations via CRISPR knock-in (as scoped).

PROTAC Treatment at Multiple Concentrations and Time Points Cells are seeded in multiwell plates and treated with a dilution series of your PROTAC compound (typically 8–10 concentrations) alongside vehicle and positive controls. Plates are incubated for defined time points (e.g., 2, 6, 24 h) to capture kinetic degradation profiles.

Cell Lysis and Target Detection Following treatment, cells are lysed under native or denaturing conditions. Residual target protein is quantified using the selected detection platform—split luciferase luminescence, TR-FRET signal, or immunoblot band intensity—with parallel viability normalization (e.g., CellTiter-Glo or MTT).

Data Analysis and Degradation Metrics Raw signals are normalized to vehicle-treated controls, and dose-response curves are fitted to calculate DC50 (half-maximal degradation concentration) and Dmax (maximal degradation percentage). Degradation kinetic plots are provided. Optionally, ternary complex formation is assessed via orthogonal binding assay.

Service Scope and Deliverables

Parameter	Typical Project Scope
Detection Platform(s)	HiBiT luminescence, TR-FRET/HTRF, Western blot, or Simple Western – selected per project goals; multiple platforms can be run in parallel.
Cell Line(s)	Mammalian lines (HEK293, HeLa, or customer-provided target-expressing line); endogenous or tagged (HiBiT/GFP) – agreed upon at project kickoff.
PROTAC Concentration Range	Typically 8–10 point serial dilution (e.g., 1 nM – 10 µM), with vehicle and reference degrader controls. Additional ranges scoped on request.
Time Points	Minimum 3 time points (e.g., 2, 6, 24 h); additional points (up to 6) can be added per project needs.
Data Output	Raw luminescence/fluorescence/chemiluminescence values, normalized degradation curves, DC50 and Dmax values, and kinetic plots (if time-course included).

Why Choose Our PROTAC Degradation Activity Detection Service?

Multiple Orthogonal Detection Platforms

We offer a menu of detection methods—split luciferase, TR-FRET, Western blot, and automated Simple Western—enabling you to select the most appropriate platform based on target accessibility, throughput needs, and budget. Having orthogonal readouts reduces the risk of false positives and provides robust cross-validation.

Experienced in PROTAC Ternary Complex Biology

Our team has deep experience with ternary complex formation assays using TR-FRET and ALPHAScreen, which help confirm that the observed degradation is driven by a cooperative target-E3 ligase interaction rather than nonspecific protein loss. This mechanistic insight is valuable for optimizing linker chemistry and ligase choice.

Flexible Assay Design for Early to Late Stage Projects

From simple endpoint degradation screening at the hit-to-lead stage to comprehensive kinetic profiling with multiple time points and counter-screens for lead optimization, we tailor the assay scope to your project’s maturity and decision gates.

Cell Line Engineering Capabilities

If your target lacks a reliable antibody or requires a tag for high-throughput detection, we can generate stable cell lines with endogenous or exogenous HiBiT tags using CRISPR knock-in. This service can be scoped separately or combined with degradation assays.

Method Spotlight

A rapid and accurate method for evaluating the degradation of pan-Akt in cells by PROTACs using NanoLuc luciferase

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Ready to Evaluate Your PROTAC Molecules?

Contact our team to discuss your project requirements and receive a customized study plan. We will work with you to select the optimal detection platform, cell line, and assay parameters to generate high-quality degradation data for your lead optimization program.

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