Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
Lab Chip. 2010 Aug 21;10(16):2046-8. doi: 10.1039/c005152g. Epub 2010 Jun 14.
Conventional biochemical analysis mainly focuses on the expression level of cellular proteins from entire cells. However, it has been increasingly acknowledged that the subcellular location of proteins often carries important information. Analysis of subcellular proteins conventionally requires subcellular fractionation which involves two steps: cell lysis to release proteins and high-speed centrifugation to separate the homogenate. Such approach requires bulky and expensive equipment and is not compatible with processing scarce cell samples of limited volume. In this study, we apply microfluidic flow-through electroporation to breach cell membranes and extract cytosolic proteins selectively in a single step. We demonstrate that this approach allows monitoring the translocation of the transcription factor NF-kappaB from the cytosol to the nucleus without the need of subcellular fractionation. Our technique is compatible with the processing of samples of various sizes and provides a simple and universal tool for bioanalytical analysis and spatial proteomics.
常规的生化分析主要关注整个细胞中细胞蛋白质的表达水平。然而，人们越来越认识到蛋白质的亚细胞位置通常携带重要信息。亚细胞蛋白质的分析通常需要亚细胞分级分离，包括两个步骤：细胞裂解以释放蛋白质和高速离心以分离匀浆。这种方法需要庞大而昂贵的设备，并且与处理有限体积的稀有细胞样品不兼容。在这项研究中，我们应用微流控流电穿孔在单个步骤中选择性地破坏细胞膜并提取细胞质蛋白质。我们证明，这种方法允许在无需亚细胞分级分离的情况下监测转录因子 NF-κB 从细胞质到细胞核的易位。我们的技术与各种大小样品的处理兼容，并为生物分析和空间蛋白质组学提供了一种简单通用的工具。
