Gosney Julie A, Ceresa Brian P
Department of Pharmacology and Toxicology, University of Louisville, 505 South Hancock St., Louisville, KY, 40202, USA.
Methods Mol Biol. 2017;1652:145-158. doi: 10.1007/978-1-4939-7219-7_11.
Scientists have separated subcellular compartments based on differences in their densities by gradient centrifugation for decades. With the proper equipment and thoughtful experimental design, density gradients are a reliable method for enriching various intracellular compartments to assess their protein composition, morphology, or biochemical activity. While variations of this basic technique have been used for separating whole cells, endosomes, synaptic vesicles, and viruses, we have found it especially useful for resolving the compartments of the endocytic pathway. In particular, this technique has been valuable for studying the regulation and signaling of the Epidermal Growth Factor Receptor (EGFR) while undergoing endocytic trafficking. In this article we detail the technical aspects of utilizing Percoll gradients to study the endocytic trafficking of the EGFR and associated proteins.
几十年来，科学家们一直通过梯度离心法，根据亚细胞区室的密度差异来分离它们。借助合适的设备和精心设计的实验，密度梯度法是富集各种细胞内区室以评估其蛋白质组成、形态或生化活性的可靠方法。虽然这种基本技术的变体已被用于分离全细胞、内体、突触小泡和病毒，但我们发现它对于解析内吞途径的区室特别有用。特别是，这项技术对于研究表皮生长因子受体（EGFR）在内吞运输过程中的调节和信号传导非常有价值。在本文中，我们详细介绍了利用Percoll梯度来研究EGFR及相关蛋白的内吞运输的技术细节。
