NanoBRET Ternary Complex Formation Assays

In a typical ternary complex system, a PROTAC or similar molecule bridges a target protein, such as a kinase, and an E3 ligase, bringing the two tagged proteins into close proximity. One protein is fused to NanoLuc luciferase, while the other is fused to HaloTag. When the third molecule promotes complex formation, the NanoLuc donor and fluorescent HaloTag ligand become closer, generating a measurable bioluminescence resonance energy transfer (BRET) signal.

The same principle can be applied to molecular glues, which promote or stabilize interactions between proteins that may not otherwise associate, and to RIPTACs, which are designed to redirect a target protein toward a second protein through induced proximity.

As ternary complex formation increases, the BRET signal becomes stronger. If the complex is destabilized or disrupted, the signal decreases. Signal intensity and concentration-response behavior can therefore be used to characterize the efficiency, potency, and dynamics of induced-proximity complex formation.

In the presence of MG-132, degradation of the target protein is inhibited. Under these conditions, a PROTAC may appear more effective at stabilizing the ternary complex, resulting in a lower apparent EC₅₀. This readout helps distinguish complex formation from subsequent target degradation and supports a more complete evaluation of induced-proximity compounds.