UBCH13 TR-FRET Assay Kit
The UbcH13 TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure UbcH13 (Ubiquitin-conjugating enzyme E2) ubiquitination activity. It utilizes biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH13 protein to complete the TR-FRET pairing. The kit contains enough purified UbcH13, purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled acceptor, and assay buffer for 400 reactions.
Figure 1: UbcH13 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 will bind 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 UbcH13 activity.
*NOTE: As of January 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.
Need us to run inhibitor screens or profile your compounds against UBCH13? Check out our Ubiquitination Screening Services or DNA Replication and Repair Screening Services.
- Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer
- Adjustable micropipettor and sterile tips
- Orbital Shaker
| Catalog # | Component | Amount | Storage |
| 80301 | UBE1 (UBA1), FLAG-Tag* | 12 µg | -80°C |
| 80323 | UBCH13 (UBE2N), His-Tag* | > 2.5 µg | -80°C |
| Biotin-Ubiquitin | 80 µl | -80°C | |
| 4 mM ATP | 1 ml | -80°C | |
| 30017 | U2 assay buffer | 2 x 10 ml | -80°C |
| 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.
UbcH13, also known as Ubiquitin Conjugating Enzyme E2N, or UBE2N, is a noncanonical ubiquitin conjugating enzyme (E2). E2 conjugating ubiquitin proteins receive Ubiquitin from Ubiquitin-activating E1 in an ATP-dependent fashion and transfer it to E3 ligases. UbcH13 has been implicated in a variety of cellular signaling processes due to its ability to catalyze the formation of lysine 63-linked polyubiquitin chains on various substrates, such as the TNF Receptor-Associated Factor 6 (TRAF6). UbcH13 is also critical for double-strand DNA repair. UbcH13 inhibition blocks NF-κB and p53 signaling in Diffuse-Large B-Cell lymphoma cell lines.
Madiraju, C., et al., J. Biomol. Screen. 2012;17(2):163-76.
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