NanoBiT Protein-Protein Interaction Assays

The NanoBiT assay uses two subunits of NanoLuc luciferase: the larger LgBiT fragment and the smaller SmBiT fragment. Each fragment is fused to one of the proteins of interest. When the two proteins interact, LgBiT and SmBiT are brought into close proximity and reconstitute an active luciferase enzyme, resulting in bioluminescence.

The intensity of the luminescent signal correlates with the strength and duration of the protein-protein interaction. Because the NanoBiT fragments have limited intrinsic affinity and are efficiently reconstituted only when brought together by the proteins of interest, the system provides low background and high interaction specificity.

The NanoBiT PPI assay measures the interaction between PDE3A and SLFN12 in the presence of increasing concentrations of DNMDP. The luminescence signal reflects reassembly of the NanoBiT luciferase fragments and provides a quantitative readout of protein-protein interaction.

GPCR/β-arrestin interactions are central to many receptor signaling pathways and provide an important application of the NanoBiT PPI platform. By fusing LgBiT to β-arrestin and SmBiT to the GPCR, receptor activation can bring the two luciferase fragments together and generate a real-time luminescent signal.

The resulting signal provides a direct cellular readout of receptor-arrestin recruitment and supports functional analysis, compound characterization, and drug discovery programs involving GPCR-targeted therapeutics.