PROTAC® Optimization Kit for PARP1-Cereblon Binding

• Background:

  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.Cereblon (CRBN) is the substrate-binding component of the E3 protein ligase complex DDB1-CUL4A-RBX1, which is involved in the ubiquitination and proteasomal degradation of target proteins. Binding of CRBN to a substrate engages its E3 ligase activity and results in the ubiquitination and ultimate degradation of the substrate. Many proteins are known targets of CRBN, including several transcription factors, growth factors and kinases. CRBN has become a target of choice for the development of many PROTACs of therapeutical interest.

• Description:

  The PROTAC® Optimization Kit for PARP1-Cereblon Binding is designed for the testing and profiling of PROTACs directed against PARP1 (Poly (ADP-ribose) -polymerase 1) and Cereblon. The PROTAC® Optimization Kit for PARP1-Cereblon Binding comes in a convenient AlphaLISA® format, with enough PARP1 Degrader iRucaparib-AP6 PROTAC®, assay buffer, recombinant purified PARP1 and CRBN (Cereblon) / DDB1 (damage specific DNA binding protein 1) /Cul4A (cullin 4A) / Rbx1 (ring-box 1) complex for 384 reactions. The PARP1 inhibitor Rucaparib is included as a control inhibitor of PROTAC binding to PARP1 (internal control) .Figure 1. Schematic representation of PARP1-Cereblon complex formation via iRucaparib-AP6 (PROTAC®) .The PROTAC® of interest is incubated with CRBN and PARP1, bringing them in close proximity. PARP1 contains a GST tag, which is recognized by the GSH donor bead. CRBN contains a FLAG tag that binds to the AlphaLISA™ acceptor bead, which is 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 interactionNeed us to run inhibitor screens or profile your compounds against PROTAC® Optimization for PARP1-Cereblon Binding? Check out ourPARP/PARPTrap™ Screening ServicesorPROTACs ® & Protein Degradation Services.

• UniProt:

  P09874

• Applications:

  Identify and optimize PROTACs targeting PARP1.Design novel molecules targeting CRBN.Directly compare the activity of different PROTACs.

• Format:

  Catalog #NameAmountStorage100329Cereblon/DDB1/Cul4A, Rbx 1 Complex*20 µg-80°C80501PARP1, GST-Tag*20 µg-80°C1 mM iRucaparib-AP6 PROTAC® (MW=887 Da) 15 µl-80°C5X PP-02 Buffer4 ml-20°C1 mM Rucaparib (MW=323 Da) 15 µl-20°C*The concentration of the proteins is lot-specific and will be indicated on the tube.

• 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:

  The final concentration of DMSO in the assay should not exceed 1%.Avoid green and blue dyes that absorb light in the AlphaScreen signal emission range (λ=520-620 nm), such as Trypan Blue.Avoid the use of 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. Media like MEM, which lacks these components, does not affect AlphaScreen assays.