TRAF6 TR-FRET Assay Kit
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.
- Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
- Adjustable micropipettor and sterile tips
| Catalog # | Name | Amount | Storage |
| 80301 | UBE1 (UBA1), Flag-Tag* | 50 µg | -80°C |
| 80314 | UBCH5b, His-Tag (Human)* | 60 µg | -80°C |
| 101597 | TRAF6, GST-Tag* | 8 µg | -80°C |
| 11236 | Ubiquitin, His-Avi Tag, Biotin-Labeled | 2 x 12 µg | -80°C |
| Tb-labeled donor | 2 x 10 µl | -20°C | |
| Dye-labeled acceptor | 2 x 10 µl | -20°C | |
| ATP (4 mM) | 2 x 1 ml | -80°C | |
| U2 Assay Buffer | 2 x 10 ml | -80°C | |
| White, nonbinding, low volume microtiter plate | Room Temp |
* The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.
Covalent conjugation of proteins to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2∼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2∼Ub to the substrate, leading to its mono- or poly-ubiquitination. TRAF6 (TNF receptor-associated factor 6) is an E3 ligase, involved in inflammation and innate immunity. Inhibitors of TRAF6 activity impact the NF-κB pathway and can prove beneficial for the treatment of auto-immune and chronic inflammatory disorders, such as psoriasis and rheumatoid arthritis.
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