CBL-B-Driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit

Catalog #
78388
$2,425 *
Size: 384 reactions
Qty
*US Pricing only. For international pricing, please contact your local distributor.
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Description

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.

CBL-B-driven Tyro3 Ubiquitination Intrachain TR-FRET

Figure 1. CBL-B-driven Tyro3 ubiquitination intrachain TR-FRET Assay Kit schematic

Synonyms
Tyrosine-protein kinase receptor, TYRO3, BYK, DTK, RSE, SKY, TIF, E3 ubiquitin-protein ligase CBL-B, Casitas B-lineage lymphoma proto-oncogene b, RING finger protein 56, RING-type E3 ubiquitin transferase CBL-B, SH3-binding protein CBL-B
Product Info
Storage and Usage
Citations
Assay Kit Format
TR-FRET
Supplied As
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.
Materials Required But Not Supplied
  • Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
  • Adjustable micropipettor and sterile tips
  • Rotating or rocker platform
Format
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.  

UniProt #
CBL-B: Q13191; TYRO-3: Q06418
Background

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.