CBL-B-Driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit
The CBL-B-driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure CBL-B E3 ligase activity in a homogeneous 384 reaction format. It utilizes a Europium-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 FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses of polyubiquitination. Of note, the assay kit does not detect mono-ubiquitination.
Alternatively, under the same experimental conditions the AXL kinase (BPS Bioscience #40180) can be used instead of Tyro3 as C-CBL ubiquitination substrate.
Figure 1. CBL-B-driven Tyro3 ubiquitination intrachain TR-FRET Assay Kit schematic
- Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
Catalog # | Name | Amount | Storage | |
80301 | UBE1 (UBA1), FLAG-tag* | 40 µg | -80°C |
Avoid multiple freeze/ thaw cycles |
80314 | UbcH5b, His-Tag* | 60 µg | -80°C | |
80415 | CBL-B, GST-tag* | 8 µg | -80°C | |
40293 | TYRO-3, GST-tag* | 16 µg | -80°C | |
78307 | TRF Ubiquitin Mix (200x) | 40 µl | -80°C | |
ATP (4 mM) | 2 x 1 ml | -80°C | ||
U2 Assay Buffer | 2 x 10 ml | -80°C | ||
79969 | White, nonbinding, low volume 384-well microtiter plate | Room 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.
Covalent conjugation to ubiquitin (Ub) is a major post-translational modifications regulating 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. This step ensures the transfer of Ub from E2∼Ub to the substrate, leading to its mono- or poly-ubiquitination.
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 and of growth factor receptor and non-receptor tyrosine kinase signaling. It contains an N-terminal tyrosine kinase binding (TKB) domain comprised of a four-helix bundle, a calcium binding EF-hand and a Src homology (SH2) domain, followed by a linker helical region and the RING domain, responsible for its catalytic function. Additionally, CBL-B contains proline-rich regions mediating the association with tyrosine- and serine phosphorylation sites, and a ubiquitin-associated (UBA)/leucine zipper domain for dimerization. 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 (Tyr) 363, which is located in the helix linker between the TKB and RING domains. Phosphorylation of Tyr363 opens CBL-B from its auto-inhibitory confirmation, allowing E2 and substrates to bind to CBL-B. CBL-B is phosphorylated for example by receptor-type tyrosine kinase Tyro3, which also serves as a substrate for CBL-B ubiquitylation both in vitro and in vivo.