PROTAC® Optimization Kit for CDK Kinase-Cereblon Binding

The PROTAC® Optimization Kit for CDK-Cereblon Binding is designed for the testing and profiling of PROTACs directed against the CDK (Cyclin-dependent kinase family) and Cereblon. The PROTAC® Optimization Kit for CDK-Cereblon Binding comes in a convenient AlphaLISA® format, with enough BSJ-03-204 PROTAC®, CDK PROTAC® buffer, purified CDK4, CDK6, and CRBN for 384 reactions. The CDK inhibitor Palbociclib is included as a control inhibitor of PROTAC binding to CDK (internal control) .Figure 1. Schematic representation of CDK-Cereblon complex formation via BSJ-03-204 PROTAC®.The PROTAC® of interest is incubated with CRBN and CDK4 or CDK6, bringing them in close proximity. CDK contains an His-tag, which is recognized by the donor bead. CRBN contains a FLAG tag that binds to the AlphaLISA™ acceptor bead, which is conjugated to 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, which emits light proportionally to the level of interaction

Product Specifications

Background

CDKs (cyclin-dependent Kinases) are a family of 20 kinases known for their role in regulating cell cycle, transcription and splicing. CDKs are organized in a pathway that safeguards the replication of DNA in each cell during cell division. The activity of CDKs is regulated by cyclins. Together CDK and cyclins, drive the cell cycle by phosphorylating other proteins. Several members of the CDK family have unique tissue specific functions and dysregulation of CDKs and their cyclin partners are associated with a range of tumor types. CDKs have emerged as potential therapeutic targets.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 with therapeutical interest.

Applications

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

Format

Catalog #ComponentAmountStorage100255Cereblon, FLAG-Tag*2 x 10 µg-80°C40104CDK4/Cyclin D3, GST-His-Tag*50 µg-80°C40206CDK6/Cyclin D3, His-Tags*30 µg-80°CBSJ-03-204 (200 μM) 2 x 20 µl-80°C3x CDK PROTAC® Buffer4 ml-20°CPalbociclib (MW=574) 120 µg-80°C*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

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, pleaseemail[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.