UC201: Gel-Based Customizable In Vitro E3 Ligase Activity Kit (Alexa647)

Ubiquitin is a small (~8.6 kDa) polypeptide that can be conjugated via its C-terminus to amine groups of lysine residues on target proteins. This conjugation is referred to as monoubiquitylation. Additional ubiquitin moieties can be conjugated to this initial ubiquitin utilizing any one of the seven lysine residues present in ubiquitin. The formation of these ubiquitin chains is referred to as polyubiquitylation. The most well characterized of this polyubiquitylation is chain formation via lysine at position 48 of ubiquitin (K48- linked chains). Monoubiquitylation has been shown to alter the localization, activity, and/or function of the target protein. The most prevalent consequence of polyubiquitylation is the proteasome-mediated degradation of the target protein. The conjugation of ubiquitin to a target protein requires the coordinated function of three distinct enzymes, E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase) resulting in isopeptide bond formation between the C-terminus of ubiquitin and the ε-amino group of the lysine residue on target proteins. Ubiquitin E3 ligases act as scaffold proteins, providing docking sites for an ubiquitin-conjugating enzyme (E2), and a target substrate. Typically, E3 ligases mediate the transfer of ubiquitin from an E2 thioester intermediate to an amide linkage with a substrate protein (Hershko and Ciechanover, 1998). In addition to the ubiquitylation of substrates, E3 ligases can also “auto ubiquitylate” themselves. This feature is utilized in in vitro assays to monitor the E3 ligase activity. This gel based In vitro ubiquitination kit has been developed to be simple, fast, and easy to use. E3 ligase assay is carried out using fluorescently labeled Ubiquitin-Alexa647 and separated on SDS-PAGE allowing researchers to directly visualize and quantify their gels saving precious time and reagents. This assay kit circumvents the more laborious and time consuming western blot-based E3 assays.

Kit Components

•  10x Assay Buffer
• Choice of E3
• Choice of E2
• Ubiquitin Activating Enzyme E1
• Ubiquitin Mix
• ATP
• Positive Control E3 with E2 (Carp2 with UBE2D3, 20x)
• 6x SDS Gel loading buffer

Info

Storage	All components should be stored at -80°C.
Stability	All components, except for the pre-coated plates, are stable for at least 2 months when stored at -80°C. Plates must be used within 30 days.

The selection of appropriate streptavidin reagent is critical to generating a robust signal. We do not recommend using avidin-horseradish peroxidase (HRP) conjugates.