DCAF1 Intrachain TR-FRET Assay Kit

Products from BPS Bioscience require a minimum order value above 400€

Applications: Screen molecules that inhibit DCAF1 E3 ligase activity in drug discovery High-Throughput Screening (HTS) applications.Determine the IC₅₀ of inhibitors of DCAF1 E3 ligase activity.Determine DCAF1 E3 ligase activity real-time kinetics.

Background: Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2ˆ¼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2ˆ¼Ub to the substrate, leading to its mono- or poly-ubiquitination.DCAF1 (DDB1 and CUL4 Associated Factor 1), also known as VprBP (Vpr binding protein), is a WD40 repeat domain-containing E3 ligase of the CRL4 (cullin RING ligase 4) subfamily of proteins. It functions as the substrate recognition unit in E3 ligase complexes, such as the EED (embryonic ectoderm development)-DDB1 (DNA damage-binding protein)-VprBP (or EDVP) and the CRL4^DCAF1 complex. The CRL4^DCAF1 complex is composed of CUL4A (cullin-4A), DDB1 and DCAF1. DCAF1 is involved in various normal physiological functions in the cell, including cell cycle regulation/progression, cell division, lipid metabolism, and miRNA biogenesis. Additionally, DCAF1 plays an E3 ligase-independent role, by acting as a kinase and phosphorylating Histone H2A. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. DCAF1 is found at high levels in cancer cells, and it is implicated in disease progression. The use of a DCAF1-BTK (Bruton's tyrosine kinase) PROTAC® resulted in degradation of BTK in cells that had acquired resistance to CRBN (cereblon)-BTK PROTACs, making DCAF1 an interesting alternative E3 ligase in cases of resistance mechanism to other commonly used PROTACs. A deeper understanding of the role of this protein and how to manipulate its multiple activities may open new therapeutic avenues.

Contraindications: This kit is compatible with up to 1% final DMSO concentration. Compounds that are fluorescent may interfere with the results, depending on their spectral excitation and emission properties.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.

Description: The DCAF1 Intrachain TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure DCAF1 (DDB1 and CUL4-associated Factor 1) complex auto-ubiquitination activity. It utilizes a mix of donor and acceptor labeled ubiquitin to complete the TR-FRET pairing. This assay measures poly-ubiquitination since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on DCAF1. The kit contains enough purified DCAF1/Rbx1 (M5I)/DDB1/CUL4A Complex, purified UBE1 and UbcH5b, detection reagents and assay buffer for 400 reactions.

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

Uniprot: Q9Y4B6

Warnings: Avoid freeze/thaw cycles

Biosafety Level: Not applicable (BSL-1)

References: Li A., et al., 2023 J Med Chem 66 (7): 5041-5060.
Schröder M., et al., 2024 Nat Commun 15 (1): 275.

• Artikelnummer: BPS82517
• Hersteller: BPS Bioscience
• Hersteller Artikelnummer: 82517
• Verpackungseinheit: 384 reactions
• Mengeneinheit: PAK