Products from BPS Bioscience require a minimum order value above 400€Applications: Screen inhibitors of C-CBL Ubiquitin ligase activity in drug discovery and HTS applicationsDetermine compound IC
50Perform C-CBL real-time kinetic analyses
Background: Covalent conjugation to ubiquitin (Ub) 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.Casitas B-lineage lymphoma (C-CBL) is the RING-type E3 ligase that that functions as a negative regulator of T cell activation. It contains an N-terminal tyrosine kinase binding domain, a SRC homology domain, and the RING domain responsible for its catalytic function. Additionally, C-CBL contains proline-rich regions mediating the association with various targets, as well as a ubiquitin-associated (UBA) domain for ubiquitin binding and dimerization. C-CBL interacts with phosphorylated proteins. C-CBL interacts with a large number of target proteins implicated in the control of cell proliferation and differentiation. The ubiquitin ligase activity of C-CBL is regulated by the phosphorylation of Tyrosine 371, which C-CBL from its auto-inhibitory conformation, allowing binding to E2 and substrates. Kinases that phosphorylate C-CBL, such as AXL, Tyro3, and SRC, also serve as substrates for ubiquitination.
Contraindications: The C-CBL-driven SRC 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.SRC kinase inhibitors may inhibit the ubiquitination reaction. It is recommended to confirm if the Test Compounds explicitly affect C-CBL ligase activity and not SRC kinase activity by determining the effect of compounds on SRC activity in the SRC Kinase Assay Kit (BPS Bioscience #79680).
Description: The C-CBL-driven SRC Ubiquitination Intrachain TR-FRET Assay Kit utilizes a Europium cryptate-labeled Ubiquitin Donor and a Cy5-labeled Ubiquitin Acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains, this assay does not detect mono-ubiquitination. The FRET-based format requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time analyses of polyubiquitination.
Figure 1: C-CBL-driven SRC ubiquitination intrachain TR-FRET Assay Kit schematic.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Supplied As: The C-CBL-driven SRC Ubiquitination Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit designed to measure the E3 ligase activity of C-CBL toward tyrosine kinase SRC in a homogeneous 384 reaction format.
Uniprot: CBL-B: Q13191; SRC: P12931
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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