Ubiquitin is a small polypeptide whose C-terminus can be conjugated to the ε-amino group of lysine residues on target proteins, a modification termed monoubiquitination. Ubiquitin itself contains seven lysine residues, allowing additional ubiquitin molecules to be sequentially linked to the already attached ubiquitin, forming polyubiquitin chains, a process known as polyubiquitination. To date, the two most extensively studied polyubiquitin chain types are those linked through lysine 48 (K48) and lysine 63 (K63). Polyubiquitination exerts diverse biological functions: K48-linked chains predominantly target proteins for proteasomal degradation, whereas K63-linked chains and others are primarily involved in non-proteolytic signaling events, such as signal transduction, DNA damage repair, vesicular trafficking, and immune regulation. This modification is reversible: deubiquitinating enzymes (DUBs) can remove ubiquitin chains, returning substrates to their unmodified state. The dynamic nature of ubiquitin modification poses a major challenge for the isolation and functional study of polyubiquitinated proteins, leaving the ubiquitination status of many proteins still poorly understood.
Based on known protein domains with ubiquitin-binding affinity, we have developed a ubiquitin-binding protein constructed by tandem fusion of domains derived from yeast DSK2 and human Ubiquilin-2 for the identification of ubiquitinated proteins. This ubiquitin-binding protein exhibits dramatically enhanced binding affinity for polyubiquitin chains compared to individual ubiquitin-associated (UBA) domains. In addition, it protects polyubiquitinated proteins, enabling detection even at relatively low abundance. These properties effectively "trap" proteins in the polyubiquitinated state. The protein binds to both K63-linked and K48-linked tetra-ubiquitin chains. Even in the absence of DUB and proteasome inhibitors, the protein provides a steric protection effect that shields polyubiquitin chains from DUB-mediated cleavage and proteasomal degradation during cell lysis and subsequent processing, thereby effectively avoiding the cellular physiological disturbances associated with chemical inhibitor supplementation in traditional methods. With its high affinity, the protein enables efficient analysis of polyubiquitinated proteins from cell, tissue, and organ samples. When equipped with affinity tags, the ubiquitin-binding protein can be used in conjunction with Western blotting for identification and characterization of ubiquitinated proteins.
Product Features:
1. Ultra-high avidity : Significantly enhanced binding to polyubiquitin chains over monomeric UBA domains, enabling efficient capture of low-abundance substrates.
2. High Sensitivity: Effectively captures trace amounts of polyubiquitinated proteins and enables stable detection of faint ubiquitination signals.
3. Precise identification: Specifically recognizes polyubiquitin chains (K48/K63, etc.), and has a stronger selective binding ability for polyubiquitin chains compared to conventional ubiquitin antibodies.
4. Native-state protection: During cell lysis, the protein protects polyubiquitinated species from degradation without the need for supplementary DUB or proteasome inhibitors. At a working concentration of 200 μg/mL, it has been validated to provide protection and enrichment efficiency comparable or superior to that achieved with inhibitor supplementation. For specific experimental requirements, inhibitors may still be added to further safeguard ubiquitin chain integrity if desired.
5. Broad downstream compatibility: Equipped with GST tag, it is compatible with Western Blot and mass spectrometry proteomics analysis.
