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Applications: Discover and optimize PROTACs targeting IRAK4Design 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. 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.IRAK4 (interleukin-1 receptor-associated kinase 4), a member of the IRAK family, is a protein kinase involved in signaling innate immune responses from Toll-like receptors. It also mediates signaling from T-cell receptors. Human and rodent genetics support the role of IRAK4 in immune function and the involvement of IRAK4-dependent signaling in certain cancers. IRAK4 protein degraders have recently entered clinical trials.
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 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™ 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.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Supplied As: This Kit comes in a convenient AlphaLISA™ format, with the IRAK4 Degrader-1 (PROTAC) added as a positive control, an optimized PROTAC Assay buffer, purified IRAK4 and CRBN proteins for 384 reactions.
Uniprot: Q9NWZ3
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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