UbcH6 TR-FRET Assay Kit
The UbcH6 TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure UbcH6 (Ubiquitin-conjugating enzyme E2) ubiquitination activity. It utilizes biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH6 protein to complete the TR-FRET pairing. The kit contains enough purified UbcH6, purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled streptavidin acceptor, and assay buffer for 400 reactions.
Figure 1: UbcH6 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 can be measured using a fluorescence plate reader capable of measuring Time Resolved-Fluorescence Resonance Energy Transfer. The TR-FRET signal is proportional to UbcH6 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 |
| 80316 | UbcH6 (UBE2E1), His-Tag* | > 2.5 µg | -80°C |
| Biotin-Ubiquitin | 80 µl | -80°C | |
| 4 mM ATP | 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.
UbcH6 (also known as Ubiquitin-conjugating enzyme E2 E1, UBE2E1) is an E2 ubiquitin-conjugating protein that receives Ubiquitin from a Ubiquitin-activating enzyme (E1) in an ATP-dependent fashion and transfers it to an E3 ligase. UbcH6 is regulated by Ubiquitin-specific protease 7 (USP7) and it is part of PRC1 (Polycomb Repressive Complex 1), an E3 complex that ubiquitinates histone H2 and it is thus involved in the proliferation of stem cells and cancer cells. It also interacts with and ubiquitinates ataxin-1, regulating its activity. Ataxin-1 is involved in spinocerebellar ataxia type 1 (SCA1), an autosomal-dominant neurodegenerative disease. Targeting UbcH6 may have therapeutical potential for the treatment of cancer and SCA1.
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