How to measure ternary complex formation for targeted protein degradation?

Flow Induced Dispersion Analysis (FIDA) measures ternary complex formation for targeted protein degradation in solution, using the Fida instrument. For bispecific degraders, which are governed by two equilibrium constants and their cooperativity, full characterisation of the biophysical parameters is possible with FIDA. Molecular glues are characterised by a single affinity constant, which simplifies the assay.

Guide to assaying ternary complex formation for targeted protein degradation using the Fida instrument

Watch the video to learn how to use the Fida instrument for assaying ternary complex formation in targeted protein degradation.

Terminology

• Indicator: Fluorescent molecules whose size is measured.
• Analyte: The compound that is titrated in the assay.

Fida experiment methods

1. Premix method: Involves pre-mixing equal amounts of indicator and analyte in vials before injection.
2. Capillary mix (Capmix) method: Employs a single indicator vial with a titration of analyte vials. The mixing happens in the capillary by the Fida instrument.
3. Capillary dissociation (CapDis) method: Involves a titration of mixed indicator and analyte vials with a single buffer vial.
4. Capillary flow (Capflex) method: Optional method, mainly for liquid phase separation and condensate analysis.

The formats are described in Which FIDA assay format should you use?

Targeted protein degradation

• Bispecific molecules: Utilised to facilitate ternary complex formation between a target protein and ubiquitin E3 ligase, leading to proteasomal degradation of the target. Governed by two equilibrium constants and their cooperativity. Full characterisation of biophysical parameters possible with Fida.
• Molecular glues: Modify the target protein surface to enhance interaction with the E3 ligase. Characterised by a single affinity constant, simplifying the assay process.

Experimental setup

• In cell lysate assays: Recommended to use the Premix method for assays directly in cell lysate. Equal amounts of analyte and indicator are mixed.
• Characterisation using bispecific molecules: Perform either a Capmix or a Premix experiment. Essential to ensure correct concentration range to avoid overshooting ternary complex formation due to the hook effect.

Data analysis using Fida software

1. Loading and evaluating data: Load data files and select the appropriate time interval for analysis.
2. Data fitting: Choose single or multispecies fitting based on the experiment. Auto mode available for efficient batch fitting of data points. Manually evaluate and correct each fit as needed.
3. Finalising analysis: Compensate for viscosity and check fluorescence areas. Select the ternary complex model in the software and input initial guesses for parameters. Apply and fit the binding curve to view the results.

The ternary complex model and the cooperativity it reports are explained in Which binding model should you use to fit FIDA affinity data?

Conclusion: This guide provides a thorough walkthrough for conducting and analysing ternary complex assays for targeted protein degradation using the Fida instrument.

What results has FIDA given for ternary complexes?

In the poster Fully automated characterization of ternary complex formation using flow induced dispersion analysis, FIDA measured the hydrodynamic radius of a labelled protein of interest as a bifunctional degrader was titrated against the protein and an E3 ligase. Binary and ternary complex formation showed as a bell-shaped binding curve, and FIDA derived the dissociation constants of the protein-degrader and degrader-E3 interactions and a cooperativity factor of 14.7, using only nanolitres of sample.

The application note Targeted Protein Degradation - Full Characterization of Ternary complexes, developed with C4 Therapeutics, shows FIDA measuring size, integrity, binding affinity and the fraction of target bound, in solution, for BiDAC degraders.

Frequently asked questions

Can FIDA measure cooperativity of a degrader?

Yes. Bispecific degraders are governed by two equilibrium constants and their cooperativity, and the ternary complex model in the Fida software reports the cooperativity together with the affinities and complex size.

Can the assay be run in cell lysate?

Yes. The Premix method is recommended for assays directly in cell lysate.

Related resources

Want to see more targeted protein degradation work with FIDA? See the poster Characterization of Molecular Glues - an automated in-solution platform, the publications Targeting the Interaction of NRF2 and β-TrCP with Molecular Glues (ACS Chemical Biology, 2026) and A degron-mimicking molecular glue drives CRBN homo-dimerization and degradation (Nature Communications, 2025), and the application note Easy and Rapid Assessment of Protein ubiquitination, e.g. in PROTAC development.

New to targeted protein degradation? The targeted protein degradation page shows the application, and What is K_D (dissociation constant)? explains the affinities. To find out whether FIDA fits your degrader programme, book a discovery call.