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South Bay Bio’s real-time TR-FRET ubiquitin conjugation assays are simple: the format is 96- or 384-well low-volume plates and assays are ‘set and forget’.
A mixture of ubiquitins labelled with cryptate and acceptor fluorophore are combined with UBA1 activating enzyme, E2 conjugating enzyme, E3 ligase, substrate protein (optional) and 10x reaction buffer, and initiated with addition of ATP. As donor and acceptor ubiquitins become incorporated into chains they come into close proximity, resulting in energy transfer (FRET) and signal detection. Readout can be collected in real time, facilitating enzyme kinetics or endpoint if preferred. Assay performance is typically unparalleled: Z’ are usually >0.8 and signal-to-noise is commonly >3000%.
The kits enable the study of auto-ubiquitination kinetics for several human recombinant E3 ligases: MDM2, ITCH and Parkin. Coupled with the assay’s short development time (as no antibody development is required for assay optimisation), the assay platform is suitable for a wide variety of academic and industry screening applications.
Kit components are also available separately.
Original Article: South Bay Bio ubiquitination TR-FRET kitsNEXT ARTICLE
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