A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

Abstract

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.