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Applications: Discover and optimize PROTACs targeting PARP1Design novel molecules targeting CRBNCompare the activities of different PROTACs
Background: CRBN (Cereblon) is the substrate-binding component of the E3 protein ligase complex DDB1-CUL4A-RBX1 involved in the ubiquitination and proteasomal degradation of target proteins. CRBN binds to DDB1 (Damaged DNA binding protein 1), to the scaffolding protein CUL4A (Cullin 4A), and its regulator RBX1 (RING-Box protein 1). Binding of CRBN to a substrate protein engages the E3 ligase activity of the complex and results in the ubiquitination and ultimate degradation of the protein substrate. Many proteins are known targets of CRBN, including several transcription factors, growth factors, kinases and more. CRBN has become a target of choice for the development of many therapeutic PROTACs.PARP1 (Poly [ADP-ribose] polymerase 1) acts as a first responder that detects DNA damage. PARP1 contributes to the efficiency of DNA repair by promoting the ADP-ribosylation of histones leading to the decompaction of chromatin structure, and by interacting with and modifying multiple DNA repair factors. PARP1 plays a role in cancer and is a well validated therapeutic target for cancer drugs.
Contraindications: Green and blue dyes, such as Trypan Blue, absorb light in the AlphaScreen™ signal emission range (520-620 nm). Avoid the use of the potent singlet oxygen quenchers such as sodium azide (NaN3) or metal ions (Fe2+, Fe3+, Cu2+, Zn2+ and Ni2+). The presence of >1% RPMI 1640 culture medium leads to a signal reduction due to the presence of excess biotin and iron in this medium. MEM, which lacks these components, does not affect AlphaScreen™ assays.
Description: 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.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.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Uniprot: P09874
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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