The UbcH5a (Ubiquitin-conjugating Enzyme E2D 1) TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET assay designed to measure UbcH5a ubiquitination activity in a 384-reaction format. It utilizes biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH5a 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 UbcH5a (full length, His-Tag), purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled acceptor, and assay buffer for 400 reactions.

Figure 1: UbcH5a TR-FRET Assay Kit schematic.
The Terbium-labeled donor will bind to the E2 conjugating protein, while the Dye-labeled acceptor will bind to Biotin-Ubiquitin. When ubiquitin is transferred to the E2 enzyme, TR-FRET occurs and can be 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.