The UbcH5c (Ubiquitin-conjugating enzyme E2 D3) TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET assay kit designed to measure UbcH5c ubiquitination activity in a 384-reaction format. It utilizes a biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH5c protein to complete the TR-FRET pairing. This FRET-based assay requires no time-consuming washing steps, making it especially suitable for high throughput screening applications. The kit contains enough purified UbcH5c (His-Tag), purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled acceptor, and assay buffer for 400 reactions.

igure 1: UbcH5c TR-FRET Assay Kit schematic.
The Terbium-labeled anti-His antibody binds to the His-tagged E2 conjugating protein, while the Dye-labeled streptavidin acceptor binds to Biotin-Ubiquitin. The complex forms when ubiquitin is transferred to the E2 enzyme, and the TR-FRET signal is measured using a fluorescence plate reader capable of measuring Time Resolved-Fluorescence Resonance Energy Transfer.

*NOTE: As of May 2023, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.