Salinas Castellanos Libia Catalina, Gatto Rodolfo Gabriel, Menchón Silvia Adriana, Blaustein Matías, Uchitel Osvaldo Daniel, Weissmann Carina
Instituto de Fisiología Biologia Molecular y Neurociencias (IFIBYNE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Fisiología, Biología Molecular y Celular (DFBMC), University of Buenos Aires (UBA), Buenos Aires 1428, Argentina.
Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA.
Membranes (Basel). 2022 Mar 31;12(4):389. doi: 10.3390/membranes12040389.
Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins and the detection of any dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved since the first use of a centrifuge to isolate organelles. In this study, we described a simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins and identify the proteins in each fraction. This method serves to identify transmembrane proteins such as channel subunits as well as PM-embedded or weakly associated proteins. This protocol uses a minute amount of cell material and typical equipment present in laboratories, and it takes approximately 3 h. The process was validated using endogenous and exogenous proteins expressed in the HEK293T cell line that were targeted to each compartment. Using a specific stimulus as a trigger, we showed and quantified the shuttling of a protein channel (ASIC1a, acid sensing ion channel) from the MO fraction to the PM fraction and the shuttling of a kinase from a cytosolic location to a nuclear location.
真核细胞中的蛋白质存在于不同的细胞区室中。许多研究需要蛋白质的特定定位以及检测细胞内蛋白质分布的任何动态变化。为此有几种方法，它们依赖于不同细胞区室的分级分离。自从首次使用离心机分离细胞器以来，分级分离方案已经不断发展。在本研究中，我们描述了一种简单的方法，该方法涉及使用台式离心机和不同的去污剂来获得富含胞质（Cyt）、质膜（PM）、膜性细胞器（MO）和核（Nu）蛋白的细胞级分，并鉴定每个级分中的蛋白质。该方法有助于鉴定跨膜蛋白，如通道亚基以及嵌入质膜或与之弱相关的蛋白质。该方案使用微量的细胞材料和实验室中常见的设备，大约需要3小时。使用靶向每个区室的HEK293T细胞系中表达的内源性和外源性蛋白质对该过程进行了验证。以特定刺激为触发因素，我们展示并量化了一种蛋白质通道（ASIC1a，酸敏感离子通道）从MO级分到PM级分的穿梭以及一种激酶从胞质位置到核位置的穿梭。
