Cullin4a/Rbx1/DDB1/CRBN – E3 Ligase Complex TR-FRET Kit

South Bay Bio’s Cullin4a/Rbx1/DDB1/CRBN Neddylated E3 Ligase TR-FRET Kit provides a fast and sensitive method monitoring ubiquitin conjugation onto the E3 complex in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto CRBN (or substrates) in a real-time detection setup, or in an end-point configuration if desired. TR-FRET uses the extended fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a short time-delay between FRET donor excitation and emission. This delay provides a means to separate “true” signal from short-lived background fluorescence, and reduce interference from compound fluorescence and other assay artifacts. The kit uses ubiquitin labeled with either Europium-Cryptate or Cyanine5 as FRET pair donor and acceptor fluorophores respectively, completely eliminating the need for antibody based detection setups. Enzymatic incorporation of the labeled ubiquitins into chains conjugated onto CRBN (or substrates) leads to an increase in fluorescence emission at 665 nm (Emacceptor) and decrease at emission wavelength 620 nm (Emdonor).