Nagl Stefan
Department of Chemistry, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, China.
Methods Mol Biol. 2019;1906:113-124. doi: 10.1007/978-1-4939-8964-5_8.
Free-flow electrophoresis (FFE) may be used for continuous and preparative separation of a wide variety of biomolecules. Isoelectric focusing (IEF) provides for the separation of compounds according to their isoelectric point (pI). Here we describe a microfluidic chip-based protocol for the fabrication, application, and optical monitoring of free-flow isoelectric focusing (FFIEF) of proteins and peptides on the microscale with optical surveillance of the microscopic pH gradient provided by an integrated pH sensing layer. This protocol may be used with modifications also for the FFIEF of other biomolecules and may serve as template for the fabrication of microfluidic chips with integrated fluorescent or luminescent pH sensor layers for FFE and other applications.
自由流动电泳（FFE）可用于多种生物分子的连续制备分离。等电聚焦（IEF）可根据化合物的等电点（pI）进行分离。在此，我们描述了一种基于微流控芯片的方法，用于在微观尺度上对蛋白质和肽进行自由流动等电聚焦（FFIEF）的制备、应用和光学监测，并通过集成pH传感层对微观pH梯度进行光学监测。该方法经修改后也可用于其他生物分子的FFIEF，并且可作为制造具有集成荧光或发光pH传感器层的微流控芯片的模板，用于FFE及其他应用。
