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.

Keywords: post-translational modification; screening; western blot.

Figure 1

Study of techniques available for detection of post-translational modifications of proteins in a medium-throughput screening (MTS) setup. (A) Analysis of advantages and disadvantages presented by dot-blot, enzyme-linked immunoabsorbent assay (ELISA) or fluorophore-linked immunosorbant assay (FLISA) and western blot (WB) regarding post-translational modifications (PTM) detection (other proteins in the lysate are represented with figures of different shapes and colors); (B) General experimental pipeline of each technique; (C) Representative images depicting the detection of mono-ubiquitylation of proliferating cell nuclear antigen (ubi-PCNA) by FLISA, dot-blot and WB and quantification of fold induction between non-irradiated (NI) and UV irradiated (UV 15 J/m²) samples displayed by each technique.

Figure 2

Characteristics of the optimization stage of the screening platform going from a classical WB towards a miniaturized WB focusing on three major areas: throughput capacity, protocol complexity and sample handling.

Figure 3

Quality controls and initial testing of the screening platform. (A) Development of stable cell line infrared fluorescent protein (iRFP)-U2OS by transfection of a iRFP-C1 plasmid and following cell sorting; (B) Assessment of the linear correlation between cell number seed and intensity detected by Odyssey equipment. The left image displays a 96-multiwell plate, where each column is an experimental replica for each condition (e.g., 12 replicas for 10,000 cells); (C) Variability and statistical parameters between different systems used for cell seeding; (D) Representative automatically-captured brightfield images depicting the range of toxicity that could be expected in the MTS. (E) Proof of concept of the screening platform with non-irradiated and UV-irradiated samples (15 J/m²) and statistical parameters of each condition.