PROTAC® Optimization Kit for BRD9-Cereblon Binding

• 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. Cereblon 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®.BRD9 (Bromodomain-containing protein 9) functions as a transcriptional regulator and is a component of a chromatin remodeling complex. It also regulates the formation of the RAD51-RAD54 complex, involved in homologous recombination. BRD9 plays a role in cancer and is a potential therapeutic target for cancer drugs.

• Description:

  The PROTAC® Optimization Kit for BRD9-Cereblon Binding is designed for the testing and profiling of PROTACs® directed against BRD9 and Cereblon (CRBN) . The BRD9 inhibitor BI-7273 is included as a control inhibitor of PROTAC® binding to BRD9. With this kit, three simple steps are required for the measurement of PROTAC® activity. First, the PROTAC® of interest is incubated with CRBN and BRD9. Next, acceptor beads are added, then donor beads, followed by reading of the Alpha-counts.Illustrationof the assay principle:a PROTAC® of interest or positive control dBRD9 (PROTAC®) interacts with both BRD9 and CRBN, bringing them in close proximity. BRD9 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.Need us to run inhibitor screens or profile your compounds against PROTAC® Optimization for BRD9-Cereblon Binding? Check out ourBromodomain Screening ServicesorPROTACs ® & Protein Degradation Services.

• UniProt:

  Q9H8M2

• Applications:

  Discover and optimize PROTACs® targeting BRD9Design novel molecules targeting CRBNCompare the activities of different PROTACs®

• Format:

  Catalog #NameAmountStorage100255FLAG-Cereblon*5 µg-80°C31091GST-BRD9*40 µg-80°C1 mM dBRD9 PROTAC® (MW=884 Da) 15 µl-80°C3x BRD9 PROTAC® Buffer**4 ml-20°C10 mM BI-7273 (MW=353 Da) 15 µl-20°C*The concentration of the proteins is lot-specific and will be indicated on the tube.**Add 30µl of 0.5 mM DTT to the assay buffer before experiment.

• Shipping Conditions:

  -80°C

• Storage Conditions:

  This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.

• Notes:

  Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor detailed troubleshooting instructions. For all further questions, please email[email protected]

• 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.