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Journal of Biomedicine and Biotechnology
Volume 2011 (2011), Article ID 421701, 8 pages
http://dx.doi.org/10.1155/2011/421701
Methodology Report

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

1Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 43210, USA
2Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA

Received 25 May 2011; Accepted 24 August 2011

Academic Editor: Xupei Huang

Copyright © 2011 Brandon J. Biesiadecki and J.-P. Jin. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.