Biesiadecki Brandon J, Jin J-P
Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 43210, USA.
J Biomed Biotechnol. 2011;2011:421701. doi: 10.1155/2011/421701. Epub 2011 Nov 13.
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.
为了解肌肉调节蛋白亚型、突变和翻译后修饰的结构-功能关系，有必要在蛋白质-蛋白质相互作用水平上探究其功能效应。评估此类蛋白质-蛋白质相互作用的传统方法既费力又需要大量纯化蛋白，而许多现有方法需要昂贵的专业设备或对蛋白质进行修饰，这可能会影响它们的相互作用。为解决这些问题，我们在近年来开发了一种基于微孔板的新型固相蛋白质结合测定方法。该方法在生理条件下评估特定的蛋白质-蛋白质相互作用，使用相对少量的蛋白质，无需对蛋白质进行修饰，也不需要专业仪器。在此，我们给出固相蛋白质结合测定的详细方法，并举例说明我们已成功应用该方法来量化肌丝调节蛋白的相互作用。我们还进一步提供了优化测定条件的注意事项及其在其他蛋白质-蛋白质相互作用研究中的更广泛应用。
