PROTAC® Optimization Kit for CDK Kinase-Cereblon Binding

Catalog #

79924

$860 *

Size: 384 reactions

Qty

Bulk/Custom

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*US Pricing only. For international pricing, please contact your local distributor.

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Description

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

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Synonyms

CRBN, AD-006, MRT2, MRT2A, Cyclin-Dependent Kinase 4, CDK4, CMM3, PSK-J3, MGC14458, Cyclin-Dependent Kinase 6, CDK6, CDKN6

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Product Data Gallery

BSJ-03-204 PROTAC®-mediated interaction of Cereblon with CDK.

Inhibition by Palbociclib or Pomalidomide of BSJ-03-204 PROTAC®-mediated interaction of Cereblon with CDK4.

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Materials Required But Not Supplied

AlphaLISA anti-FLAG acceptor beads, 5 mg/ml (PerkinElmer #AL112C)
Alpha Nickel donor beads, 5 mg/ml (PerkinElmer #AS101D)
Optiplate 384 (PerkinElmer #6007290)
AlphaScreen microplate reader
Adjustable micropipettor and sterile tips

Format

Catalog #	Component	Amount	Storage
100255	Cereblon, FLAG-Tag*	2 x 10 µg	-80°C
40104	CDK4/Cyclin D3, GST-His-Tag*	50 µg	-80°C
40206	CDK6/Cyclin D3, His-Tags*	30 µg	-80°C
	BSJ-03-204 (200 μM)	2 x 20 µl	-80°C
	3x CDK PROTAC® Buffer	4 ml	-20°C
	Palbociclib (MW=574)	120 µg	-80°C

*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

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.

References

Brand M., et al., 2019 Cell Chem. Biol. 26(2):300-306.
Nunes J., et al., 2019 ACS Med Chem Lett 10(7): 1081-1085.
Łukasik P., et al., 2021 Int J Mol Sci. Mar 13;22(6):2935.

Storage/Stability

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

Applications

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

Shipping Temperature

-80°C

Notes

Troubleshooting Guide
Visit bpsbioscience.com/assay-kits-faq for 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.

Warnings

BSJ-03-204 is a thalidomide-derivative, which is known to cause severe birth defects in humans. It is very important to use all appropriate precautions when handling this compound. Avoid freeze/thaw cycles

Disclaimers and Limitations

BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.

End-users should immediately report any issues to [email protected] upon receipt of the kit, such as missing components, damaged vials, no dry ice, etc.

Scientific Category

PROTACs

Regulatory Status

Research Use Only