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Applications: Screen inhibitors of C-CBL Ubiquitin ligase activity in drug discovery and high throughput applications.Determine compound IC50 and perform C-CBL real-time kinetic analyses.
Background: Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications regulating 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 E2~Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2~Ub to the substrate, leading to its mono- or poly-ubiquitination.Casitas B-lineage lymphoma (C-CBL) is a RING-type E3 ligase and a member of the CBL family of proteins that comprises three homologues. It contains an N-terminal tyrosine kinase binding (TKB) domain comprised of a four-helix bundle, a calcium binding EF-hand and a Src homology (SH2) domain, followed by a linker helical region and the RING domain responsible for its catalytic function. Additionally, C-CBL contains proline-rich regions and several tyrosine-phosphorylation sites mediating the association with various targets, as well as a ubiquitin-associated (UBA) domain for ubiquitin binding and dimerization. C-CBL interacts with a large number of target proteins implicated in cell survival, migration and proliferation. The ubiquitin ligase activity of C-CBL is up-regulated by the phosphorylation of Tyrosine (Tyr) 371, which is located in the helix linker between the TKB and RING domains. Phosphorylation of Tyr371 opens C-CBL from its auto-inhibitory conformation, allowing binding to E2 and substrates. C-CBL is phosphorylated by receptor-type tyrosine kinases such as Tyro3, which can also serve as a substrate for C-CBL-mediated ubiquitination.
Contraindications: The C-CBL-driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 5% DMSO solution in buffer and using 4 µl per well.Tyro3 kinase inhibitors may inhibit the ubiquitination reaction. It is recommended to confirm if the Test Compounds explicitly affect C-CBL ligase activity and not Tyro3 kinase activity by determining the effect of compounds on Tyro3 activity in the TYRO3 Kinase Assay Kit ( #79593).
Description: The C-CBL-driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure C-CBL E3 ligase activity in a homogeneous 384 reaction format. It utilizes a Europium cryptate-labeled Ub (donor) and a Cy5-labeled Ub (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for high throughput applications as well as real-time analyses of polyubiquitination. Of note, the assay kit does not detect mono-ubiquitination.Alternatively, under the same experimental conditions the AXL kinase (BPS Bioscience #40180) can be used instead of Tyro3 as C-CBL ubiquitination substrate.
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)
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