DCAF11 Intrachain TR-FRET Assay Kit
The DCAF11 Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit designed to measure DCAF11 auto-ubiquitination activity in a homogeneous 384-reaction format. It utilizes Europium-labeled Ubiquitin (donor) and Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. This assay measures poly-ubiquitination since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on DCAF11. As a homogenous assay, it requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time analyses.
Figure 1. DCAF11 Intrachain TR-FRET Assay Kit Schematic.
Need us to run inhibitor screens or profile your compounds against DCAF11? Check out our Ubiquitination Screening Services or DNA Replication & Repair Screening Services.
- Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
| Catalog # | Name | Amount | Storage |
| 80301 | UBE1 (UBA1), FLAG-Tag (E1)* | 50 µg | -80°C |
| 80314 | UbcH5b, His-Tag (E2)* | 60 µg | -80°C |
| 101495 | DCAF11/Rbx1/CUL4B/DDB1 complex, (E3)* | 62 µg | -80°C |
| 78307 | TRF Ubiquitin Mix (200x) | 40 µl | -80°C |
| ATP (4 mM) | 2 x 1 ml | -80°C | |
| CBL assay buffer 2 | 2 x 10 ml | -80°C | |
| 79969 | White, nonbinding Corning, low volume 384-well microtiter plate | Room Temp |
* The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.
The Ubiquitin Mix is sourced from South Bay Bio LLC.
Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2∼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2∼Ub to the substrate, leading to its mono- or poly-ubiquitination.
DDB1 and CUL4-associated Factor 11 (DCAF11) is a protein that associates with CUL4A or CUL4B to form a complex with E3 ligase activity. DCAF11 interacts with and ubiquitinates nuclear factor E2-related factor 2 (Nrf2), which is known to regulate cellular stress response genes, including those associated with oxidative stress. Additionally, DCAF11 activity is repressed by transcriptional factor EB (TFEB), allowing for stabilization of proteins such as Nrf2. Because Nrf2 and TFEB are involved in misfolded protein clearance, and oxidative stress is known to contribute to diseases such as Alzheimer’s, Huntington’s, and Parkinson’s disease, DCAF11 is a potential target for the treatment of multiple neurodegenerative disorders. Like most E3 ligases, DCAF11 ubiquitinates itself.
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