DESCRIPTION

• Shiptemp: -80°C (dry ice)
• Warnings: Avoid freeze/thaw cycles.
• Category: Ubiquitination
• 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 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.
• Description: The ChoosE3-Freedom Intrachain TR-FRET Assay Kit contains E1 and E2 enzymes, ATP, an optimized TRF Ubiquitin Mix, and a universal buffer. Purified E3 ligase MDM2 is also provided as an internal quality control. The assay was designed with 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 formed on the E3 ligase, the assay measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics.
• Methodology: TR-FRET
• Supplied As: The ChoosE3-Freedom Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET assay kit, designed to measure the auto-polyubiquitination of any purified E3 ligase of interest in a homogeneous 384 reaction format.
• Unspsc Code: 41121811
• Unspsc Name: Laboratory microchemistry kits
• Applications: Measure the auto-polyubiquitination of any E3 ligase of interest Identify novel E3 ligases or variants of known ligases Screen inhibitors or activators of an E3 ligase of interest in HTS applications Determine compound IC50 Perform E3 ligase real-time analyses
• Product Type: Assay Kit
• Biosafety Level: Not applicable (BSL-1)
• Contraindications: The intrachain TR-FRET Assay Kit is not suitable for measuring mono-ubiquitination. Weak signals may be obtained for multi-mono-ubiquitination or for short poly-ubiquitin chains. This kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 5% DMSO solution in assay buffer and using 4 µl per well. The E3 ligase used in the assay should be purified. The assay will not perform well when using cell lysates due to the presence of other E3 ligases and ubiquitin.
• Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
• Scientific Category: Ubiquitination