PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains

Catalog #

82291

$2,050 *

Size: 384 reactions

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

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Description

The PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains is designed for the testing and profiling of PROTACs targeting the bromodomains on the BET (bromodomain and extraterminal) protein family. After tertiary complex formation between the E3 protein, the PROTAC and the protein of interest, ubiquitination of the target protein is a critical step preceding its degradation. The PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains comes in an AlphaLISA® format with enough optimized assay buffer, purified recombinant E1 (UBE1), E2 (UbcH5b), and E3 enzymes (Cereblon complex), BRD3 (BD2) (bromodomain-containing protein 3) (amino acids 305-417) as target protein, ATP, and biotinylated ubiquitin for 384 reactions. This kit also contains the PROTAC dBET1 as the control, and JQ1 as control inhibitor.

Figure 1. Schematic representation of the principle of the PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains.

dBET1 or a PROTAC® of interest is incubated with Cereblon complex and BRD3 (BD2), bringing them in close proximity. Ubiquitination reaction occurs following the addition of E2 enzyme, ATP, biotinylated ubiquitin, and, finally, E1 enzyme. Once ubiquitination has taken place, AlphaLISA® donor and acceptor beads are added. BRD3 (BD2) contains a GST-tag, which is recognized by GSH-AlphaLISA™ acceptor beads. Ubiquitinated BRD3 (BD2) contains biotin-ubiquitin, that binds to Streptavidin donor beads. Upon excitation of the donor bead, a singlet oxygen is generated. The singlet oxygen excites the acceptor bead, which emits light. The resulting signal is directly proportional to the level of ubiquitination of BRD3 (BD2).

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Product Data Gallery

dBET1 and dBET6 PROTAC-Driven BRD3 (BD2) ubiquitination, two similar PROTACS with different linker sizes

Inhibition of dBET1 mediated ubiquitination of BRD3 (BD2) by Cereblon in the presence of various inhibitors

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Materials Required But Not Supplied

AlphaLISA GSH Acceptor Beads, 250 µg (PerkinElmer #AL109C)
AlphaScreen Streptavidin-Conjugated Donor Beads, 5 mg/ml (PerkinElmer #6760002S)
Optiplate 384 (PerkinElmer #6007290)
AlphaScreen microplate reader
Adjustable micropipettor and sterile tips

Format

Catalog #	Name	Amount	Storage
80301	UBE1 (UBA1), FLAG-Tag*	25 µg	-80°C
80314	UbcH5b, His-Tag (Human)*	60 µg	-80°C
100329	Cereblon/DDB1/Cul4A/ Rbx1 Complex*	60 µg	-80°C
31033	BRD3 (BD2), GST-Tag*	5 µg	-80°C
	dBET1 (MW=785 Da)	2 x 0.31 µg	-20°C
27403	10 mM (+)-JQ1	10 µl	-20°C
	U2 Assay Buffer	10 ml	-20°C
	10 mM ATP	400 ml	-80°C
	Biotin-Ubiquitin	400 ml	-80°C
	Plate sealer	1	Room Temp

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

Background

Cereblon is the substrate-binding component of the E3 protein ligase complex Cereblon-CUL4A (cullin-4A)-RBX1 (RING-box protein 1) that targets proteins for degradation through the proteasome system. The binding of Cereblon to a substrate protein engages the E3 ligase activity of the complex and results in the ubiquitination and ultimate degradation of the substrate protein. PROTACs are bifunctional molecules that bring together a therapeutic target and an E3 ligase to promote the ubiquitination and ultimately the degradation of the target. Efficiency of ubiquitination is an important factor for optimization of heterobifunctional degraders. BRD3, also known as RING3L (RING3-like protein) belongs to the BET family of proteins and is involved in transcription by associating with acetylated lysines present in histones and transcription factors. Chromosomal translocations of BRD3 with NUT (nuclear protein in testis) can lead to cancer, and it has been shown that the use of BET inhibitors or depletion of BRD3 can slow down cancer progression in models of prostate cancer. The use of PROTAC technology for degradation of BRD proteins is an active field of research. Development of novel PROTAC molecules involved in the recruitment of BRD3 (BD2) provides a framework for future efforts to utilize Cereblon to degrade other proteins of interest.

References

Kaur, S.D., et al. 2023 Cancer Lett; 556: 216065.
Wang C., et al., 2022 J Enzyme Inhib Med Chem 37(1): 1694-1703.

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 functional activity of PROTACs targeting bromodomains of the BET family.
Design novel molecules linking bromodomains of the BET family and the Cereblon complex.
Directly compare ubiquitination of target proteins by different PROTACs.
Identify potential inhibitors of ubiquitination.

Shipping Temperature

-80°C

Notes

Safety
This product is for research purposes only and not for human or therapeutic use. This product should be considered hazardous and is harmful by inhalation, in contact with skin, eyes, clothing, and if swallowed. If contact occurs, wash thoroughly. dBET1 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.

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

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

Ubiquitination

Regulatory Status

Research Use Only

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PROTAC-Driven Ubiquitination Assay Kit for BET Bromodomains

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