ChoosE3-Freedom™ Intrachain TR-FRET Assay Kit
The ChoosE3-Freedom Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET assay kit, designed to measure the auto-polyubiquitination of any purified E3 ligase of interest in a homogeneous 384 reaction format. The ChoosE3-Freedom Intrachain TR-FRET Assay Kit contains E1 and E2 enzymes, ATP, an optimized TRF Ubiquitin Mix, and a universal buffer. Purified E3 ligase MDM2 is also provided as an internal quality control.
The assay was designed with a Europium-labeled Ubiquitin donor and a Cy5-labeled Ubiquitin acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on the E3 ligase, the assay measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics.
Figure 1: ChoosE3-Freedom Intrachain TR-FRET Assay Kit schematic.
Need us to run inhibitor screens or profile your compounds against ChoosE3-Freedom™? 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 | Avoid multiple freeze/ thaw cycles |
| 80314 | UbcH5b, His-Tag* | 60 µg | -80°C | |
| 100409 | MDM2, GST-Tag* | 10 µg | -80°C | |
| 78307 | TRF Ubiquitin Mix (200x) | 50 µl | -80°C | |
| ATP (4 mM) | 2 x 1 ml | -80°C | ||
| U2 Assay Buffer | 2 x 10 ml | -80°C | ||
| White, nonbinding Corning, low volume 384-well 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 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.
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