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

• 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 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.

• 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.Need us to run inhibitor screens or profile your compounds against C-CBL-Driven SRC Ubiquitination? Check out ourUbiquitination Screening Services.

• UniProt:

  CBL-B: Q13191; SRC: P12931

• Applications:

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

• Format:

  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.

• Shipping Conditions:

  -80°C

• Storage Conditions:

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

• Notes:

  Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor detailed troubleshooting instructions. For all further questions, please email[email protected]

• 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) .