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

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

Product Specifications

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 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.

UniProt

CBL-B: Q13191; SRC: P12931

Applications

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

Format

Catalog #NameAmountStorage80301UBE1 (E1) *40 µg-80°C80314UBCH5b (E2) *60 µg-80°C80415CBL-B, 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.The Ubiquitin Mix is sourced from South Bay Bio LLC.

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 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) .