CUL1 Intrachain TR-FRET Assay Kit

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

Applications:

• Screen molecules that inhibit CUL1 Ub ligase activity in drug discovery high throughput screening (HTS) applications.
• Determine compound IC
• Perform CUL1 real-time kinetic studies.

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.CUL1, or cullin 1, is part of an E3 ligase complex, the SCF (SKP1 (s-phase kinase associated protein 1)- CUL1- F-box protein) complex, that also involves SKP2 and RBX1 (RING-box protein 1). The SCF complex is involved in the ubiquitination of proteins that act on cell cycle, signal transduction and transcription, with CUL1 serving as a structural scaffold. Cullins can be used in the context of targeted protein degradation in cancer therapy.

Contraindications: The CUL1 Intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration.

Description: The CUL1 Intrachain TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure CUL1 (cullin 1) auto-ubiquitination. It utilizes a Europium-labeled ubiquitin (Ub) donor as well as Cy5-labeled Ub acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on CUL1, this assay measures poly-ubiquitination. The kit comes in a convenient 384-well format and contains enough purified CUL1/SKP1/SKP2/RBX1 Complex, purified UBE1 (ubiquitin-like modifier activating enzyme 1), UbcH5b (Ubiquitin-conjugating enzyme E2 D2), Ubi-Mix™, ATP, and assay buffer for 400 reactions.

Figure 1. CUL1 Intrachain TR-FRET Assay Kit schematic.
E1 and E2 enzymes, Europium cryptate-labeled Ubiquitin (TR-FRET donor) and Cy5-labeled Ubiquitin (TR-FRET acceptor) are incubated with CUL1 complex. The donor and acceptor are incorporated into poly-ubiquitin chains formed on CUL1, allowing energy transfer to occur. The TR-FRET signal is proportional to CUL1 activity

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

Warnings: Avoid freeze/thaw cycles

Biosafety Level: Not applicable (BSL-1)

References: Sweeney M., et al., 2020 Scientific Reports 10:13942.

• Artikelnummer: BPS82219
• Hersteller: BPS Bioscience
• Hersteller Artikelnummer: 82219
• Verpackungseinheit: 384 reactions
• Mengeneinheit: PAK