UBCH5a TR-FRET Assay Kit
The UbcH5a TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure UbcH5a (Ubiquitin-conjugating Enzyme E2D 1) ubiquitination activity. It utilizes biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH5a protein to complete the TR-FRET pairing. The kit contains enough purified UbcH5a, 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. The TR-FRET signal is proportional to UbcH5a 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
- Orbital Shaker
| Catalog # | Component | Amount | Storage |
| 80301 | UBE1, FLAG-Tag* | 12 µg | -80°C |
| 80315 | UBCH5a (UBE2D1), His-Tag* | > 2.5 µg | -80°C |
| Biotin-Ubiquitin | 80 µl | -80°C | |
| ATP (4 mM) | 1 ml | -80°C | |
| U2 Assay Buffer | 2 x 10 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.
UbcH5a (also known as Ubiquitin-conjugating Enzyme E2D 1, UBE2D1) is an E2 conjugating ubiquitin protein, able to bind ubiquitin noncovalently in other surfaces besides the active site. Ubiquitination is a critical multistep pathway involved in cellular homeostasis. E2 conjugating ubiquitin proteins serve as a link between E1 ubiquitin activating enzymes and E3 ligases, by receiving a ubiquitin from E1, in an ATP dependent mode, and transferring it to E3. UbcH5a is closely related to SFT (Stimulator of Iron Transport) and is involved in the iron dependent ubiquitination of HIF (Hypoxia-Inducible Factor). It is overexpressed in the liver of patients with hereditary hemochromatosis. It also functions in the ubiquitination of the tumor-suppressor protein p53. Inhibition of UbcH5 may become a promising target in cancer therapy.
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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