Screening tools for CBL activity from BPS Bioscience

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This news was updated in August 2024

Assay kits to measure CBL ubiquitination activity

CBL-B and c-CBL are E3-ubiquitin ligases that function as intracellular checkpoints by targeting proteins for degradation via the proteasomal pathway. CBL-B (CBL stands for Casitas B-lineage lymphoma proto-oncogene) specifically functions as a negative effector of T cell activation by promoting the downregulation of the TCR (T cell receptor). A mutation in the CBL-B gene has been linked to type 1 diabetes and potentially other autoimmune diseases. C-CBL interacts with receptor tyrosine kinases involved in cell signaling and activation, targeting them for degradation and acting in a negative feedback loop to deactivate these receptors.

The first step in the process of ubiquitination involves the binding of ubiquitin (Ub) to an E1 Ub-activating enzyme. This step requires ATP hydrolysis to activate ubiquitin and forms a thioester bond between the E1 enzyme and ubiquitin. In the second step, ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2), where Ub is also linked via a thioester bond. Finally, Ub is transferred from the Ub-E2 complex to a lysine residue in a substrate by an E3 Ub ligase such as CBL.

Measuring the enzymatic activity of CBL, therefore, requires the presence of ubiquitin, E1 and E2 enzymes, purified CBL, and ATP, all components supplied in the kits. BPS Bioscience’s options of CBL-B and c-CBL assay kits use TR-FRET to measure CBL auto-ubiquitination.

Alternatively, TR-FRET intrachain assay kits containing CBL substrates Tyro3, SRC or AXL,  provide additional insights on substrate poly-ubiquitination.

TR-FRET assay kit advantages

• Homogeneous (no-wash) assays, ideal for high-throughput applications
• High sensitivity and low background
• Real-time readings do not require equilibrium conditions, compatible with kinetic measurements
• Available in 384-well plate format, use small amounts of materials including test compounds
• All components and protocols included with the kits

TR-FRET assay kits to measure CBL-B and c-CBL auto-ubiquitination

These assays are designed to measure c-CBL or CBL-B auto-ubiquitination activity in a homogeneous (no-wash) 384-well format. Only poly-ubiquitination, and not mono-ubiquitination, is measured. The assays use biotin-labeled ubiquitin and a terbium-labeled anti-GST antibody that targets GST-tagged c-CBL or CBL-B to complete the TR-FRET pairing. No time-consuming washing steps are required, making them especially suitable for high throughput screening applications to identify effective inhibitors of c-CBL and CBL-B.

Tyro3 ubiquitination intrachain TR-FRET assay kits

The c-CBL and CBL-B-driven Substrate Ubiquitination Intrachain TR-FRET Assay kits are sensitive, high-throughput-friendly TR-FRET assays designed to measure c-CBL or CBL-B E3 ligase activity towards a kinase substrate in a 384-well format. They use a Europium cryptate-labeled Ub (donor) and a Cy5-labeled Ub (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains, these assays allow real-time analyses of poly-ubiquitination, and do not detect mono-ubiquitination.

Resources
Research poster: Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity
eBook: TR-FRET assay kits simplify and accelerate drug discovery