Fig. (1A) Principle of a TR-FRET-based Nef/SH3 protein-protein interaction assay. The quaternary complex of His-Nef, GST-SH3, α-GST Eu-labeled mAb, and α-His XL665-labeled mAb generates the proximity for a FRET pair between the donor lanthanide fluorophor europium and acceptor fluorophor XL665. The Eu chelates emit light at 620 nm after excitation at 340 nm and thereby excite neighboring XL665 FRET acceptors. Excited XL665 decays under the emission of photons with a wavelength of 665 nm. The 665 nm emission or the 665/620 nm ratio is proportional to the number of quaternary complexes formed. Excited Eu chelates have a long half-life. The time-resolved detection minimizes interference with extrinsic fluorescence. (B) Schematic of the resonant waveguide grating (RWG)-based assay for monitoring proteinligand or protein-protein interactions in real time. Here, the target protein His-Nef (or off-target GST-SH3) is immobilized on the preactivated aldehyde surface of an optical biosensor whose photonic crystal composition allows the reflection of broadband light only in a narrow range of wavelengths, the peak wavelength value (PWV). Binding of a chemical (compound) or biological (protein) ligand to His-Nef results in a change of the refractory index of the biosensor, measured as a shift of the peak wavelength value (ΔPWV) that is proportional to the change in mass on the surface.