UBCH5c TR-FRET Assay Kit
The UbcH5c TR-FRET Assay Kit is a homogeneous, sensitive TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay kit designed to measure UbcH5c (Ubiquitin-conjugating enzyme E2 D3) ubiquitination activity. It utilizes a biotin-labeled Ubiquitin and a Terbium-labeled antibody recognizing the His-tagged UbcH5c protein to complete the TR-FRET pairing. The kit contains enough purified UbcH5c, purified UBE1, Biotin-Ubiquitin, anti-His Tb-labeled donor, dye-labeled acceptor, and assay buffer for 400 reactions.
Figure 1: UbcH5c 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 UbcH5c 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 UBCH5c? Check out our Ubiquitination Screening Services.
- 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 (UBA1), FLAG-Tag* | 12 µg | -80°C |
| 80313 | UbcH5c, His-Tag (Human)* | > 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 | |
| 79969 | 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.
UbcH5c (also known as Ubiquitin-conjugating enzyme E2 D3, UBE2D3) is an E2 conjugating ubiquitin protein of the UbcH5 protein family. E2 conjugating ubiquitin proteins receive Ubiquitin from E1 in an ATP-dependent manner and transfer it to E3. UbcH5c regulates the ubiquitination of transcription factors such as nuclear factor kappa-B (NF-κB) and p53 tumor suppressor, and it thus involved in diseases such as rheumatic arthritis (RA) and cancer. Small molecule inhibitors of UbcH5c alter expression levels and activity of p53 and are thus promising therapeutical candidates 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.
Powered by Bioz
San Diego, CA 92121
858-202-1401
Products are for Research Use Only
COMPANY
Copyright © 2024
BPS Bioscience, Inc.
All rights reserved.
Diagrams on this website are created with BioRender.com.