Cereblon Intrachain TR-FRET Assay Kit
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.
The Cereblon (CRBN) protein via its interaction with the DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B), and regulator of Cullins 1 (RoC1) forms the functional E3 Ub ligase complex. In this complex, Cereblon functions as a substrate receptor that mediates the ubiquitination and subsequent proteasomal degradation of target proteins. Cereblon complex is involved in many biological processes including cell proliferation and apoptosis. That is why it has been employed for targeted protein degradation in the treatment of cancer. Like most E3 ligases, Cereblon complex ubiquitinates itself and this auto-ubiquitination promotes its Ub ligase activity.
The Cereblon intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure Cereblon auto-ubiquitination activity in a homogeneous 384 reaction format. It utilizes a Europium cryptate-labeled Ub (donor) as well as Cy5-labeled Ub (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on Cereblon, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses of polyubiquitination.
Figure 1. Cereblon intrachain TR-FRET Assay Kit schematic
Need us to run inhibitor screens or profile your compounds against Cereblon? 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* | 50 µg | -80°C |
| 80314 | UbcH5b, His-Tag* | 60 µg | -80°C |
| 100329 | Cereblon/DDB1/Cul4A/Rbx1 Complex* | 60 µg | -80°C |
| 78307 | TRF Ubiquitin Mix (200x) | 40 µl | -80°C |
| ATP (4 mM) | 2 x 1 ml | -80°C | |
| U2 Assay Buffer | 2 x 10 ml | -80°C | |
| 79969 | White, nonbinding, low volume microtiter plate | 1 | 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.
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