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. 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.

Figure 1: ChoosE3-Freedom Intrachain TR-FRET Assay Kit schematic.