Data: Real-Time Bio-Layer Interferometry Ubiquitination Assays as Alternatives to Western Blotting

Abstract

Ubiquitination is a crucial cellular pathway enabling normal cellular functions such as cell cycle regulation, DNA damage repair, cell signaling, and maintenance of protein homeostasis. However, abnormalities or failures in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases including cancers, neurodegenerative diseases, immune disorders, and metabolic disorders. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. However, screening and development of small molecule inhibitors that inhibit the activity of enzymes within the ubiquitination cascade is challenging due to the lack of knowledge of the complex dynamics and regulatory mechanisms of the cascade. Thus, the detection of ubiquitination is important for enabling a better understanding of the molecular mechanisms behind the ubiquitination cascade. 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 1-second resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously and result in substantial time savings. In addition, the reagent concentrations and quantities are similar to those used by traditional western blot-based ubiquitination assays. Thus, the developed BLI ubiquitination assay is introduced to the field as a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.