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

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 E2∼Ub 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.

Screen inhibitors of C-CBL Ubiquitin ligase activity in drug discovery and HTS applicationsDetermine compound IC50Perform C-CBL real-time kinetic analyses

Catalog #NameAmountStorage80301UBE1 (E1) *40 µg-80°C80314UBCH5b (E2) *60 µg-80°C100370C-CBL, GST-Tag*8 µg-80°C40484SRC, His-Tag*12 µg-80°C78307TRF Ubiquitin Mix (200x) 40 µl-80°CATP (4 mM) 2 x 1 ml-80°CU2 Assay Buffer2 x 10 ml-80°C79969White, nonbinding, low volume 384-well microtiter plateRoom Temp*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

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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) .