UBCH5b TR-FRET Assay Kit
The Ubch5b TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure UbcH5b (Ubiquitin-conjugating enzyme E2 D2) ubiquitination activity. It utilizes biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged Ubch5b protein to complete the TR-FRET pairing. The kit contains enough purified UbcH5b, purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled acceptor, and assay buffer for 400 reactions.
Figure 1: UbcH5b 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. The TR-FRET signal is proportional to UbcH5b activity.
*NOTE: As of January 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.
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- Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
| Catalog # | Component |
Amount |
Storage |
| 80301 | UBE1 (UBA1), FLAG-Tag* | 12 µg | -80°C |
| 80314 | UBCH5b, full length, His-Tag (Human)* | > 2.5 µg | -80°C |
| Biotin-Ubiquitin | 80 µl | -80°C | |
| 4 mM ATP | 1 ml | -80°C | |
| U2 Assay Buffer | 2x10 ml | -80°C | |
| 30017 | Anti-His Tb-labeled donor | 10 µl | -20°C |
| Dye-labeled acceptor | 10 µl | -20°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.
UbcH5b (also known as Ubiquitin-conjugating Enzyme E2 D2, UBE2D2) is an E2 conjugating ubiquitin protein. E2 conjugating proteins receive Ubiquitin from E1 in an ATP dependent fashion and transfer it to an E3 ligase. UbcH5b is overexpressed in certain cancer types. It functions in the ubiquitination of tumor-suppressor proteins p53 and p62. UbcH5b-p62 complex confers therapeutic resistance in triple negative breast cancer cells. UbcH5b is also involved in endocytosis-mediated degradation of MHC class I molecules, and overexpression has been found in inflammatory bowel disease. Inhibitors of UbcH5b may prove beneficial in cancer and auto-immune disease treatment.
1. Saville, M.K., et al., J. Biol. Chem. 2004; 279(40): 42169-81.
2. Kim, J.H., et al., BMB Rep. 2015; 48(1): 25-29.
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