Journal
ANALYST
Volume 147, Issue 15, Pages 3434-3443Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/d2an00577h
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Funding
- National Key R&D Program of China [2020YFE0202200, 2017YFA0505002, 2017YFA0505100, 2017YFA0505700]
- National Natural Science Foundation of China [32071431, 31901037, 31870824, 91839302, 32070668]
- Innovation Foundation of Medicine [19SWAQ17, 20SWAQX34, AWS17J008]
- CAMS Innovation Fund for Medical Sciences [2019-I2M-5-017]
- Beijing-Tianjin-Hebei Basic Research Cooperation Project [J200001]
- Foundation of State Key Lab of Proteomics [SKLP-K201704, SKLP-K201901]
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The study introduces a new method TUF-WB+ that significantly improves the robustness and sensitivity of polyubiquitination signal detection, while also reducing the experimental operation time. The ThUBD-HRP probe is capable of conducting in situ polyubiquitination detection in cells, providing a powerful tool for ubiquitination signal detection.
Polyubiquitination signal deliver diverse cellular signal, which have been recognized as a sophisticated ubiquitin code. The perception and transduction of ubiquitination signal depend on the specificity and sensitivity of the ubiquitin-binding domain. Accurate and sensitive detection of polyubiquitination signal is crucial for revealing the dynamic cellular ubiquitin-regulated events. Western blotting (WB) and immunohistochemistry (IHC) are the most widely used biochemical strategies to detect ubiquitination signal on substrates under diverse physiological and pathological conditions. However, anti-ubiquitin antibodies fail to reflect polyubiquitination signal unbiasedly because of their strong preference for K63-linked ubiquitin chains. Herein, we demonstrated that our previously developed tandem hybrid ubiquitin-binding domain (ThUBD) chemically labeled with a reporter group such as horseradish peroxidase (ThUBD-HRP) could significantly improve the robustness and sensitivity of polyubiquitination signal detection. This advanced method was named TUF-WB Plus (TUF-WB+). The TUF-WB+ method significantly increases the sensitivity and accuracy of ubiquitin detection and requires a shorter experimental operation time. Furthermore, it enables the ThUBD-HRP probe to function as a powerful tool for spatial in situ polyubiquitination detection in cells by immunohistochemistry. Our newly developed ThUBD-HRP probe and TUF-WB+ method provide a robust and powerful tool for ubiquitination signal detection with hypersensitivity in an unbiased manner.
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