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Background: Covalent conjugation of proteins 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. TRAF6 (TNF receptor-associated factor 6) is an E3 ligase, involved in inflammation and innate immunity. Inhibitors of TRAF6 activity impact the NF- κB pathway and can prove beneficial for the treatment of auto-immune and chronic inflammatory disorders, such as psoriasis and rheumatoid arthritis.
Contraindications: The TRAF6 intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration.TRAF6, GST-Tag (BPS Bioscience #101597) is comprised of amino acids 51-159. Inhibitors targeting TRAF6 regions outside of the 51-159 amino acid region cannot be assessed using this assay kit.
Description: The TNF receptor associated factor 6 (TRAF6) TR-FRET Assay Kit is a sensitive TR-FRET Assay Kit, designed to measure TRAF6 (amino acids 51-159) auto-ubiquitination activity in a homogeneous 384 reaction format. This assay measures mono- or poly-ubiquitination. As a homogeneous assay, it requires no time-consuming washing steps and is especially suitable for high throughput screening (HTS) applications as well as real-time analyses. Read out requires a fluorescent plate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET).The assay takes advantage of biotinylated ubiquitin, which is linked to TRAF6 during auto-ubiquitination. The streptavidin-conjugated acceptor binds to ubiquitin-biotin while a Terbium-conjugated anti-GST antibody donor binds to GST-tagged TRAF6, allowing TR-FRET pairing. The resulting TR-FRET signal is directly proportional to TRAF6 mono or poly-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.
Supplied As: The kit contains enough purified UBE1 (E1), UBCH5b (E2), E3 ligase TRAF6 (amino acids 51-159, GST-tag), Biotin-Ubiquitin, labelled donor and acceptor, ATP, and U2 Assay Buffer for 384 reactions.
Uniprot: Q9Y4K3
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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