Fluorescein-labeled ThUBD probe for super-sensitive visualization of polyubiquitination signal in situ cells

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the ubiquitin and ubiquitin chains. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to their functions. We demonstrated that an artificial tandem hybrid UBD (ThUBD) exhibits an unbiased high affinity to all ubiquitin chains and is a promising tool for global ubiquitination profiling research. In this study, we labeled fluorescein on the four cysteine residues in the N-terminal glutathione S-transferase (GST) tag of ThUBD, generating a fluorescein-labeled ThUBD (ThUBD-Flu) probe for direct polyubiquitination signal imaging and visualization. Compared to the canonical ubiquitin antibody method, the ThUBD-Flu is hyper-sensitive and accurate to detect ubiquitination signal. More importantly, the ThUBD-Flu probe provided, for the first time, a widely applicable, super-sensitive and unbiased technique for in situ detection of intracellular polyubiquitination signal through immunofluorescence staining, which was only achievable with recombinant fluorescence tag fused ubiquitin gene previously. We propose that ThUBD-Flu, combined with evolving microscopy technology, could serve as prototypes to track and trace cellular polyubiquitination signal in vivo.