The E1 LITE – UBE1 Activity Assay Kit is based on FRET between donor and acceptor fluorophores associated with the two ubiquitin molecules bound to the activated UBE1 ternary complex (see figure below). The assay employs streptavidin terbium as the donor fluorophore, which is directed to the complex by biotinylated ubiquitin and fluorescein labeled ubiquitin as the acceptor fluorophore. The long-lived fluorescence of the terbium donor allows a time-delayed reading of the FRET signal, or time-resolved FRET (TR-FRET), which reduces the contribution of short-lived background emissions resulting from from buffers, proteins, and chemical compounds. The assay can be run in a 96-well or 384-well format and produces a reproducible signal with a Z’ > 0.7. The kit contains sufficient reagents for one 1/2-volume 96-well plate.

“The time resolved FRET assay for E1 ternary complex formation represents a significant advance in speed and utility that should be particularly useful for those interested in routine quality control measurements, high throughput activity screens, or rapid reaction kinetics for which isotopic methods are precluded.”

– Arthur L. Haas, Ph.D.Roland Coulson Professor and HeadDepartment of Biochemistry and Molecular BiologyLSU Health Sciences Center School of Medicine

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