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 schematicNeed us to run inhibitor screens or profile your compounds against MURF3 Intrachain? Check out ourUbiquitination Screening Services.

Product Specifications

Background

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.

UniProt

UBE1: P22314; UBCH5b: P62837; MURF3: Q9BYV2

Applications

Screen molecules that inhibit MURF3 Ub ligase activity in HTS applicationsDetermine compound IC50Perform MURF3 real-time kinetic analyses.

Format

Catalog #NameAmountStorage80301UBE1 (UBA1), FLAG-tag (E1) *50 µg-80°CAvoid multiple freeze/ thaw cycles80314UbcH5b, His-Tag (E2) *60 µg-80°C81054MURF3, SUMO-His-tags (E3) *10 µg-80°C78307TRF Ubiquitin Mix (200x) 50 µl-80°CATP (4 mM) 2 x 1 ml-80°CU2 Assay Buffer2 x 10 ml-80°CWhite, nonbinding Corning, low volume microtiter plateRoom Temp

Shipping Conditions

-80°C

Storage Conditions

This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.

Contraindications

The MURF3 intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 5% DMSO solution in buffer and using 4 µl per well.