文章基本信息

标题：Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations
本地全文：下载
作者：Jessica S. Sadick ; Molly E. Boutin ; Diane Hoffman-Kim 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2016
卷号：6
期号：1
DOI：10.1038/srep33999
语种：English
出版社：Springer Nature
摘要：Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.