Journal ArticleDOI
Chemically labeled ThUBD permits rapid and super-sensitive imaging of polyubiquitination signals.
Weidi Xiao,Shuai Huang,Yuan Gao,Tong Liu,Naikang Li,Xinyi Sui,Yonghong Wang,Guan Yang,Lei Chang,Xianjiang Kang,Changzhu Duan,Weijie Qin,Ping Xu,Yanchang Li +13 more
TLDR
It is demonstrated that the previously developed tandem hybrid ubiquitin-binding domain (ThUBD) chemically labeled with a reporter group such as horseradish peroxidase (Thubi-HRP) could significantly improve the robustness and sensitivity of polyubiquitination signal detection.Abstract:
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.read more
Citations
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Journal ArticleDOI
Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7.
TL;DR: In this article , the authors compared the efficiency and effectiveness of substrate screening from the entire proteomics to the ubiquitinomics filter using yeast deubiquitinating enzyme, Ubp7.
References
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