A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.
A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.
复制标题
用于生物传感器验证和检查影响生物传感器活性的刺激的高内涵测定。
DOI：
10.1002/0471143030.cb1415s65
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发表时间：
2014
影响因子：
--
通讯作者：
Hahn,KlausM
中科院分区：
文献类型：
--
作者：
Slattery,ScottD;Hahn,KlausM
生物传感器是用于监测体内许多不同蛋白质行为的有价值的工具。对新生物传感器的需求很高，但它们的开发和表征可能很困难。在生物传感器的设计过程中，有必要评估不同的生物传感器结构对特异性、亮度和荧光响应的影响。通过将生物传感器与刺激或抑制生物传感器报告的活性的上游蛋白质共表达，可以确定生物传感器的最大激活和失活状态之间的差异，并检查对特定蛋白质的响应。我们在此描述了一种使用自动显微镜在96孔板格式中进行生物传感器验证的方法。该方案产生剂量响应曲线，能够有效检查许多参数，并且与细胞悬浮液测定不同，允许目视检查（例如，用于细胞健康和生物传感器或调节器定位）。解决了单链和双链Rho GTdR生物传感器的优化，但该测定适用于可以在贴壁细胞中表达或以其他方式加载的任何生物传感器。该测定还可用于生物传感器验证以外的目的，使用良好表征的生物传感器作为上游分子效应的读数。© 2014年由John Wiley & Sons，Inc.
Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co‐expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96‐well plate format using an automated microscope. This protocol produces dose‐response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single‐chain and dual‐chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well‐characterized biosensor as a readout for effects of upstream molecules. © 2014 by John Wiley & Sons, Inc.