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Applications: Screen molecules that inhibit TRAF6 Ub ligase activity in drug discovery HTS applicationsDetermine compound IC50Perform TRAF6 real-time kinetics
Background: Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that 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 E2ˆ¼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2ˆ¼Ub to the substrate, leading to its mono- or poly-ubiquitination.
Contraindications: The TRAF6 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.
Description: The TRAF6 intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure TRAF6 auto-ubiquitination activity in a homogeneous 384 reaction format. It utilizes a Europium-labeled ubiquitin (Ub) donor as well as Cy5-labeled Ub acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on TRAF6, this assay measures poly-ubiquitination. As a homogeneous assay, it requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time analyses.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Uniprot: Q9Y4K3
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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