WWP1 Intrachain TR-FRET Assay Kit
The WWP1 intrachain TR-FRET Assay Kit utilizes a Europium cryptate-labeled Ubiquitin (donor) as well as 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 WWP1, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetic analyses of polyubiquitination.
Figure 1. WWP1 intrachain TR-FRET Assay Kit schematic
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- 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 |
| 80405 | WWP1, FLAG-Tag* | 45 µg | -80°C |
| 78307 | 200x Ubi-Mix™ | 40 µl | -80°C |
| 4 mM ATP | 2 x 1 ml | -80°C | |
| U2 Assay Buffer | 2 x 10 ml | -80°C | |
| 79969 | White, nonbinding Corning, low volume microtiter plate | Room Temp |
*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.
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.
WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1) is a HECT-type E3 Ub ligase belonging to the NEDD4 family of E3 ligases. WWP1 interacts with and ubiquitinates many substrates, including transcription factor ΔNp63 as well as phosphatase and tensin homolog (PTEN), and regulates the degradation of sodium channels and membrane receptors. Genetic mutations or regulatory defects involving WWP1 are associated with neurological disorders and cancer. Aberrant expression of WWP1 in gastric, prostate, and breast cancer, for instance, is an area of high interest, and therefore, WWP1 represents an excellent candidate for targeting in multiple cancer types.
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