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TR-FRET Cereblon-4C Binding Assay Kit
E3 ubiquitin ligases are a large family of enzymes that catalyze the critical final step in the ubiquitination cascade. Cereblon (CRBN) serves as the substrate-recognition component of the E3 ubiquitin ligase complex CRBN/DDB1/CUL4A/RBX1 (CRBN-4C). CRBN is one of the most widely utilized E3 ligases in the design of PROTACs (Proteolysis-Targeting Chimeras) for targeted protein degradation (TPD) in drug discovery.
The TR-FRET CRBN Binding Assay Kit is designed to measure the binding affinity between CRBN and its ligands. The kit includes Tag1-CRBN-4C (a complex of CRBN/CUL4A/DDB1/RBX1), a terbium-labeled anti-Tag1 antibody, and a fluorescently labeled CRBN ligand, thalidomide. When CRBN binds to thalidomide, the terbium donor (on the anti-Tag1 antibody) is brought into close proximity to the fluorescent acceptor (fluorophore-labeled thalidomide), resulting in fluorescence resonance energy transfer (FRET). The binding interaction can be quantitatively measured as an HTRF signal, calculated from the ratio of the emission intensities at 665 nm (acceptor) and 620 nm (donor). If a test compound competes with thalidomide for CRBN binding, the HTRF signal will decrease, indicating inhibition of CRBN–thalidomide interaction.
