MDM2 Intrachain TR-FRET Assay

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 human Mouse double minute 2 homolog (MDM2) is an E3 Ub ligase and the master regulator of tumor suppressor proteins such as p53. Thus, high activity of MDM2 can promote tumor formation by targeting tumor suppressor proteins for proteasomal degradation, enabling cancer cell survival and proliferation. That is why MDM2 is an attractive potential drug target in cancer immunotherapy. Like most E3 ligases, MDM2 ubiquitinates itself and this auto-ubiquitination stimulates MDM2 Ub ligase activity. The MDM2 intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure MDM2 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 MDM2, 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.