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 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. The von Hippel-Lindau protein (VHL) interacts with Elongins B and C, Cullin 2, and Ring Box Protein 1 (Rbx1) to form the functional E3 Ub ligase complex where it functions as a substrate recognition entity. Most of the tumor-derived mutations within VHL disrupt its tumor suppressor role by compromising its substrate receptor function and generally whole VHL complex Ub ligase activity. VHL complex not only targets the alpha subunits of the heterodimeric transcription factor hypoxia inducible factor (HIF) for ubiquitylation and proteasomal degradation, but it is involved in many other biological processes related to tumor growth. That is why it is an attractive potential drug target in cancer immunotherapy. Like most E3 ligases, VHL complex can ubiquitinate itself. The VHL intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure VHL auto-ubiquitination activity in a homogeneous 384 reaction format. It utilizes a Europium cryptate-labeled 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 VHL, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses of polyubiquitination.
