The TNF receptor associated factor 6 (TRAF6) TR-FRET Assay Kit is a sensitive TR-FRET Assay Kit, designed to measure TRAF6 (amino acids 51-159) auto-ubiquitination activity in a homogeneous 384 reaction format. This assay measures mono- or poly-ubiquitination. As a homogeneous assay, it requires no time-consuming washing steps and is especially suitable for high throughput screening (HTS) applications as well as real-time analyses. Read out requires a fluorescent plate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET).

Figure 1. TRAF6 TR-FRET Assay Kit schematic.
The assay takes advantage of biotinylated ubiquitin, which is linked to TRAF6 during auto-ubiquitination. The streptavidin-conjugated acceptor binds to ubiquitin-biotin while a Terbium-conjugated anti-GST antibody donor binds to GST-tagged TRAF6, allowing TR-FRET pairing. The resulting TR-FRET signal is directly proportional to TRAF6 mono or poly-ubiquitination.