UbcH7 TR-FRET Assay Kit
The UbcH7 TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure UbcH7 (Ubiquitin-conjugating enzyme E2 L3 or UBE2L3) ubiquitination activity. It utilizes biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH7 protein to complete the TR-FRET pairing. The kit contains enough purified UbcH7, purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled streptavidin acceptor, and assay buffer for 400 reactions.
Figure 1: UbcH7 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 Biotinylated-Ubiquitin. The complex forms when ubiquitin is transferred to the E2 enzyme, and the TR-FRET signal can be measured using a fluorescence plate reader capable of measuring Time Resolved-Fluorescence Resonance Energy Transfer. The TR-FRET signal is proportional to UbcH7 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 # | Name | Amount | Storage |
| 80301 | UBE1 (UBA1), FLAG-Tag* | 12 µg | -80°C |
| 80317 | UbcH7, His-Tag (E. coli-Derived)* |
> 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.
UbcH7 (Ubiquitin-conjugating enzyme E2 L3 or UBE2L3) is an E2 ubiquitin-conjugating protein that receives Ubiquitin from a Ubiquitin-activating (E1) enzyme and subsequently interacts with a Ubiquitin ligase (E3) to conjugate Ubiquitin to substrate proteins and mediate their selective degradation. UbcH7 is involved in the ubiquitination of the transcription factors NF-κB, p53, and c-Fos, and is involved in cell cycle regulation. Abnormalities in UbcH7 activity have been linked to autoimmune diseases such as rheumatoid arthritis, celiac disease, Crohn's disease, and systemic lupus erythematosus. UbcH7 is, therefore, an attractive therapeutic target.
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