MDM2 E3 Ligase TR-FRET Kit

South Bay Bio’s MDM2 E3 Ligase TR-FRET Kit provides a fast and sensitive method monitoring ubiquitin conjugation onto GST-MDM2 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 MDM2 in a real-time detection setup, or in an end-point configuration if desired. TR-FRET uses the extended fluorescence emission decay lifetimes 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 reduces 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 MDM2 leads to an increase in fluorescence emission at 665 nm (Emacceptor) and decrease at emission wavelength 620 nm (Emdonor).