The PROTAC® Optimization Kit for PARP1-Cereblon Binding is designed for the testing and profiling of PROTACs® directed against PARP1 and Cereblon (CRBN). This Kit comes in a convenient AlphaLISA™ format, with the PARP1 Degrader iRucaparib-AP6 (PROTAC®) added as positive control, PARP1 buffer, purified PARP1 and CRBN proteins for 384 reactions. The PARP1 inhibitor Rucaparib is included as a control that blocks PROTAC® binding to PARP1. With this kit, three simple steps are required for the measurement of PROTAC® activity. First, the PROTAC® of interest is incubated with CRBN and PARP1. 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 iRucaparib-AP6 (PROTAC®) interacts with both PARP1 and CRBN, bringing them in close proximity. PARP1 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. The singlet oxygen 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.