MURF3 Intrachain TR-FRET Assay Kit
The MURF3 intrachain TR-FRET Assay Kit 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 MURF3, 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: MURF3 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 (E1)* | 50 µg | -80°C | Avoid multiple freeze/ thaw cycles |
| 80314 | UbcH5b, His-Tag (E2)* | 60 µg | -80°C | |
| 81054 | MURF3, SUMO-His-tags (E3)* | 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 microtiter plate | Room Temp | |||
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.
Muscle-Specific RING Finger Protein 3 (MURF3, also known as TRIM54) is a protein that associates with microtubules in cardiac and skeletal muscle cells, where it is involved in myoblast differentiation and microtubule network development. MURF3 is known to interact with and ubiquitinate filamin, which plays critical roles in actin filament formation and cytoskeletal structure. MURF3 mutations are associated with myopathies and cardiomyopathies, and therefore MURF3 is a potential target for the treatment of these diseases.
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