Development and Optimization of a Miniaturized Western Blot-Based Screening Platform to Identify Regulators of Post-Translational Modifications

Abstract

Post-translational modifications (PTMs) are fundamental traits of protein functionality and their study has been addressed using several approaches over the past years. However, screening methods developed to detect regulators of PTMs imply many challenges and are usually based on expensive techniques. Herein, we described the development and optimization of a western blot-based platform for identification of regulators of a specific PTM—mono-ubiquitylation of proliferating cell nuclear antigen (PCNA). This cell-based method does not require specific equipment, apart from the basic western blot (WB) devices and minor accessories, which are accessible for most research labs. The modifications introduced to the classical WB protocol allow the performance of PTM analysis from a single well of a 96-well plate with minimal sample manipulation and low intra- and inter-plate variability, making this method ideal to screen arrayed compound libraries in a 96-well format. As such, our experimental pipeline provides the proof of concept to design small screenings of PTM regulators by improving the quantitative accuracy and throughput capacity of classical western blots.

Endoplasmic reticulum sequestration empowers phosphorylation profiling on the yeast surface

“…Currently, techniques such as Western blotting, reverse phase protein arrays, and mass spectrometry are considered the standard for identification of posttranslational modifications in cellular systems. [11][12][13][14][15] In each of these approaches, cells are exposed to stimuli to elicit a signaling response, followed by lysis and downstream analysis of cell-wide modifications made to proteins. A combination of these methods was required to discover lysine malonylation, a significant posttranslational modification.…”