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

Catalog #

78821

$2,310 *

Size: 384 reactions

Qty

Bulk/Custom

Add to Favorites

*US Pricing only. For international pricing, please contact your local distributor.

●

Purchase

●

Description

The CBL-B-driven AXL 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 AXL ubiquitination intrachain TR-FRET Assay Kit schematic.

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

●

Synonyms

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, Signal transduction protein CBL-B, CBLB, RNF56, Nbla00127, UFO, AXL oncogene, Tyro

●

Product Data Gallery

Inhibition of CBL-B-driven AXL ubiquitination

Product Info

Storage and Usage

Citations

Assay Kit Format

TR-FRET

Supplied As

The CBL-B-driven AXL ubiquitination intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit designed to measure the E3 ligase activity of CBL-B toward tyrosine kinase AXL 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
80314	UbcH5b, His-Tag *	60 µg	-80°C
80415	CBL-B, GST-Tag*	8 µg	-80°C
100174	AXL, FLAG-Tag*	10 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; AXL: P30530

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.

Storage/Stability

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

Applications

Screen inhibitors of CBL-B Ubiquitin ligase activity in drug discovery and HTS applications
Determine compound IC₅₀
Perform real-time kinetic analyses

Shipping Temperature

-80°C

Notes

Troubleshooting Guide
Visit bpsbioscience.com/assay-kits-faq for detailed troubleshooting instructions. For all further questions, please email [email protected]

Contraindications

The CBL-B-driven AXL 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.

AXL kinase inhibitors may inhibit the ubiquitination reaction. It is recommended to confirm if the Test compounds explicitly affect CBL-B ligase activity and not AXL kinase activity by determining the effect of compounds on AXL activity in the AXL Kinase Assay Kit (BPS Bioscience#79711).

Warnings

Avoid freeze/thaw cycles

Disclaimers and Limitations

BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.

End-users should immediately report any issues to [email protected] upon receipt of the kit, such as missing components, damaged vials, no dry ice, etc.

Scientific Category

Ubiquitination

Regulatory Status

Research Use Only

Skip to the end of the images gallery

Skip to the beginning of the images gallery

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

PRODUCTS

SERVICES

RESOURCES

COMPANY