Department of Chemistry and Biochemistry, 651 East High Street, Miami University, Oxford, OH, 45056, United States.
Department of Chemistry and Biochemistry, 651 East High Street, Miami University, Oxford, OH, 45056, United States.
Anal Biochem. 2023 Oct 15;679:115296. doi: 10.1016/j.ab.2023.115296. Epub 2023 Aug 20.
Ubiquitination is a crucial cellular pathway enabling normal cellular functions. Abnormalities in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases. Efforts to screen and develop small molecule inhibitors targeting portions of the ubiquitination cascade require rapid and robust methods for detecting ubiquitination. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides one-second time resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously, and reagent concentrations are identical to those used by traditional western blot-based ubiquitination assays. The developed BLI ubiquitination assay is a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.
泛素化是一种至关重要的细胞途径，能够实现正常的细胞功能。泛素化过程的异常可能导致细胞功能障碍，并引发一系列疾病。筛选和开发针对泛素化级联反应特定部分的小分子抑制剂的努力需要快速而强大的方法来检测泛素化。在该领域，人们已经做出了巨大的努力，使用各种技术包括荧光、分光光度法、化学发光、NMR 和放射性示踪剂来检测泛素化。检测泛素化最常用的方法是蛋白质印迹法。然而，蛋白质印迹法耗时且难以用于寻求精细时间过程实验。在这里，我们展示了使用生物层干涉测量法实时快速检测泛素化。已经将 E3 连接酶自身泛素化系统和底物泛素化测定作为新开发测定的测试。所开发的 BLI 泛素化测定提供了一秒的时间分辨率，并直接在生物传感器结合的靶标上检测多聚泛素链的形成。结果立即返回，并且试剂浓度与传统基于蛋白质印迹的泛素化测定中使用的试剂浓度相同。所开发的 BLI 泛素化测定是一种可行的替代传统蛋白质印迹测定的方法，可用于快速实时检测泛素化。
