CBL-B-Driven SRC Ubiquitination Intrachain TR-FRET Assay Kit

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Applications: Screen inhibitors of CBL-B Ubiquitin ligase activity in drug discovery and HTS applicationsDetermine compound IC₅₀Perform 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 proto-oncogene-b (CBL-B) is the RING-type E3 ligase 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, CBL-B contains proline-rich regions mediating the association with phosphorylated proteins, and a dimerization domain. CBL-B interacts with a large number of target proteins implicated in the control of cell proliferation, differentiation, and cell morphology. The ubiquitin ligase activity of CBL-B is up-regulated by the phosphorylation of Tyrosine 363, which opens CBL-B from its auto-inhibitory conformation, allowing binding of E2 and substrates. Kinases that phosphorylate CBL-B, such as AXL, Tyro3, and SRC, also serve as substrates for ubiquitination.

Contraindications: The CBL-B-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 CBL-B ligase activity and not SRC kinase activity by determining the effect of compounds on SRC activity in the SRC Assay Kit (BPS Bioscience#79680).

Description: The CBL-B-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: CBL-B-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 CBL-B-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 CBL-B 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)

• Artikelnummer: BPS78820
• Hersteller: BPS Bioscience
• Hersteller Artikelnummer: 78820
• Green Labware: Nein
• Verpackungseinheit: 384 rxns.
• Mengeneinheit: PAK