PROTAC(R) Optimization Kit for IRAK4-Cereblon Binding

The PROTAC Optimization Kit for IRAK4-Cereblon Binding is designed for the testing and profiling of PROTACs directed against IRAK4 and Cereblon (CRBN). The IRAK4 ligand-1 is included as a control that blocks PROTAC binding to IRAK4. With this kit, only three simple steps are required for the measurement of PROTAC activity. First, the PROTAC of interest is incubated with CRBN and IRAK4. Next, acceptor beads are added, then donor beads, followed by reading of the Alpha-counts. Illustration of the assay principle: A PROTAC of interest or positive control IRAK4 Degrader-1 (PROTAC) interacts with both IRAK4 and CRBN, bringing them in close proximity. IRAK4 contains a GST-tag, recognized by the GSH donor bead, while CRBN contains a FLAG-tag that binds to the AlphaLISA(TM) acceptor bead conjugated with an anti-FLAG antibody. Upon excitation of the donor bead, a singlet oxygen is generated by the donor bead, which excites the acceptor bead and emits light proportionally to the level of interaction. AlphaLISA(TM) immunoassays are a no-wash alternative to ELISA immunoassays. These assays are robust and ideal for a minimal hands-on approach.