Ma Guangzhong, Wan Zijian, Wang Shaopeng
Biodesign Center for Biosensors and Bioelectronics, Arizona State University, Tempe, USA.
School of Electrical, Computer and Energy Engineering, Arizona State University, Tempe, USA.
Bio Protoc. 2021 Mar 5;11(5):e3934. doi: 10.21769/BioProtoc.3934.
Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of single-molecule techniques has shown great potential in improving the resolution of the traditional protein analysis methods to the single-molecule level. However, such single-molecule techniques measure either size or charge, and it is challenging to measure both at the same time. Recently, we have developed a single-molecule approach to address this problem. We tether the single proteins to a surface with a polymer linker and drive them into oscillation with an electric field. By tracking the electromechanical response of the proteins to the field using an optical imaging method, the size and charge can be obtained simultaneously. Binding of antibodies or ions to the tethered protein also changes the size and charge, which allows us to probe the interactions. This protocol includes fabrication of protein oscillators, configuration of the optical detection system, and analysis of the oscillation signal for quantification of protein size and charge. We wish this protocol will enable researchers to perform comprehensive single-protein analysis on a single platform.
电泳和蛋白质印迹法是蛋白质研究中用于蛋白质检测和鉴定的重要工具。这些传统技术基于大小和电荷差异分离蛋白质，并通过抗体结合来鉴定蛋白质。在过去十年中，单分子技术的出现显示出将传统蛋白质分析方法的分辨率提高到单分子水平的巨大潜力。然而，此类单分子技术要么测量大小，要么测量电荷，要同时测量两者具有挑战性。最近，我们开发了一种单分子方法来解决这个问题。我们用聚合物连接体将单个蛋白质 tether 到表面，并用电场驱动它们振荡。通过使用光学成像方法跟踪蛋白质对电场的机电响应，可以同时获得大小和电荷。抗体或离子与 tethered 蛋白质的结合也会改变大小和电荷，这使我们能够探测相互作用。该方案包括蛋白质振荡器的制造、光学检测系统的配置以及对振荡信号进行分析以定量蛋白质的大小和电荷。我们希望该方案能使研究人员在单个平台上进行全面的单蛋白质分析。
