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Chemically labeled ThUBD permits rapid and super-sensitive imaging of polyubiquitination signals
Analyst ( IF 3.6 ) Pub Date : 2022-07-07 , DOI: 10.1039/d2an00577h Weidi Xiao 1 , Shuai Huang 1 , Yuan Gao 2 , Tong Liu 1 , Naikang Li 3 , Xinying Sui 4 , Yonghong Wang 5 , Guan Yang 1 , Lei Chang 1 , Xianjiang Kang 3 , Changzhu Duan 4 , Weijie Qin 1 , Ping Xu 1, 3, 5, 6, 7 , Yanchang Li 1, 3
Analyst ( IF 3.6 ) Pub Date : 2022-07-07 , DOI: 10.1039/d2an00577h Weidi Xiao 1 , Shuai Huang 1 , Yuan Gao 2 , Tong Liu 1 , Naikang Li 3 , Xinying Sui 4 , Yonghong Wang 5 , Guan Yang 1 , Lei Chang 1 , Xianjiang Kang 3 , Changzhu Duan 4 , Weijie Qin 1 , Ping Xu 1, 3, 5, 6, 7 , Yanchang Li 1, 3
Affiliation
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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.
中文翻译：
化学标记的 ThUBD 允许对多泛素化信号进行快速和超灵敏的成像
多泛素化信号传递多种细胞信号，这些信号已被公认为复杂的泛素代码。泛素化信号的感知和转导取决于泛素结合域的特异性和敏感性。多泛素化信号的准确和灵敏检测对于揭示动态的细胞泛素调节事件至关重要。蛋白质印迹 (WB) 和免疫组织化学 (IHC) 是最广泛使用的生化策略，用于在不同的生理和病理条件下检测底物上的泛素化信号。然而，抗泛素抗体不能公正地反映多泛素化信号，因为它们强烈偏爱 K63 连接的泛素链。在此处，我们证明了我们之前开发的串联杂交泛素结合域 (ThUBD) 用报告基团如辣根过氧化物酶 (ThUBD-HRP) 化学标记可以显着提高多泛素化信号检测的稳健性和灵敏度。这种先进的方法被命名为 TUF-WB Plus (TUF-WB+ )。TUF-WB +方法显着提高了泛素检测的灵敏度和准确度，需要更短的实验操作时间。此外，它使 ThUBD-HRP 探针能够作为一种强大的工具，通过免疫组织化学在细胞中进行空间原位多泛素化检测。我们新开发的 ThUBD-HRP 探针和 TUF-WB +方法为泛素化信号检测提供了强大而强大的工具，并且以无偏的方式具有超敏性。
更新日期：2022-07-07
中文翻译：
化学标记的 ThUBD 允许对多泛素化信号进行快速和超灵敏的成像
多泛素化信号传递多种细胞信号，这些信号已被公认为复杂的泛素代码。泛素化信号的感知和转导取决于泛素结合域的特异性和敏感性。多泛素化信号的准确和灵敏检测对于揭示动态的细胞泛素调节事件至关重要。蛋白质印迹 (WB) 和免疫组织化学 (IHC) 是最广泛使用的生化策略，用于在不同的生理和病理条件下检测底物上的泛素化信号。然而，抗泛素抗体不能公正地反映多泛素化信号，因为它们强烈偏爱 K63 连接的泛素链。在此处，我们证明了我们之前开发的串联杂交泛素结合域 (ThUBD) 用报告基团如辣根过氧化物酶 (ThUBD-HRP) 化学标记可以显着提高多泛素化信号检测的稳健性和灵敏度。这种先进的方法被命名为 TUF-WB Plus (TUF-WB+ )。TUF-WB +方法显着提高了泛素检测的灵敏度和准确度，需要更短的实验操作时间。此外，它使 ThUBD-HRP 探针能够作为一种强大的工具，通过免疫组织化学在细胞中进行空间原位多泛素化检测。我们新开发的 ThUBD-HRP 探针和 TUF-WB +方法为泛素化信号检测提供了强大而强大的工具，并且以无偏的方式具有超敏性。
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