Besnard Julien, Okumoto Sakiko
Virginia Tech.
Virginia Tech;
J Vis Exp. 2014 Jul 31(89):e51657. doi: 10.3791/51657.
Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories. One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.
基因编码传感器能够在亚细胞分辨率下对生物分子进行实时监测。在过去15年里,出现了种类繁多的此类生物分子传感器,其中一些已成为许多实验室常规使用的不可或缺的工具。基因编码传感器令人兴奋的应用之一是将这些传感器用于研究细胞转运过程。转运蛋白的动力学和底物特异性等特性可在细胞水平上进行研究,为细胞类型特异性分析转运活性提供了可能。在本文中,我们将以基因编码谷氨酰胺传感器为例,展示如何观察转运蛋白动力学。我们将讨论实验设计、实验设置的技术细节以及实验后分析的注意事项。