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.

Figure 1. 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™ 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™ immunoassays are a no-wash alternative to ELISA immunoassays. These assays are robust and ideal for a minimal hands-on approach.