Ubi-Trans(TM) UBA6-UbcH5b TR-FRET Assay Kit

The Ubi-Trans(TM) UBA6-UbcH5b TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay designed to measure UBA6 (ubiquitin-like modifier activating enzyme 6)-dependent transfer of Ub (ubiquitin) to UbcH5b (Ubiquitin-conjugating enzyme E2 D2) E2 enzyme. It utilizes a Terbium (Tb)-labeled donor and dye-labeled streptavidin acceptor to complete the TR-FRET pairing. The kit comes in a convenient 384-well format and contains enough purified UbcH5b, purified UBA6, Biotin-Ubiquitin, anti-His Tb (terbium)-labeled donor, dye-labeled streptavidin acceptor, and assay buffer for 400 reactions. Figure 1: Ubi-Trans(TM) UBA6-UbcH5b TR-FRET Assay Kit schematic. In the presence of ATP, UBE6 (E1) transfers biotin-conjugated ubiquitin to the substrate UbcH5b (E2). The Tb-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 by E1. In the absence of ubiquitination, the complex does not form and energy transfer does not occur. When E2 is ubiquitinated the complex is formed, TR-FRET pairing is complete, and an increase in acceptor emission is observed. Thus, TR-FRET signal is proportional to UBE6 activity.