Subcellular Fractionation of Cultured Human Cell Lines

Subcellular localization is crucial for the proper functioning of a protein. Deregulation of subcellular localization may lead to pathological consequences and result in diseases like cancer. Immuno-fluorescent staining and subcellular fractionation can be used to determine localization of a protein. Here we discuss a protocol to separate the nuclear, cytosolic, and membrane fractions of cultured human cell lines using a centrifuge and ultracentrifuge. The membrane fraction contains plasma membranes and ER-golgi membranes, but no mitochondria or nuclear structures. The fractions can be further analyzed using Western blotting. This protocol is based on that from Dr. Richard Patten at Abcam, and was modified and utilized in a publication by Huang et al. (2012)., 亚细胞定位对于蛋白质的正常功能是至关重要的。 亚细胞定位的失调可能导致病理结果并导致诸如癌症的疾病。 免疫荧光染色和亚细胞分级分离可用于确定蛋白质的定位。 在这里我们讨论使用离心机和超速离心机分离培养的人类细胞系的核，细胞溶质和膜部分的协议。 膜级分含有质膜和ER-高尔基体膜，但没有线粒体或核结构。 可以使用蛋白质印迹法进一步分析级分。 该协议基于来自Abcam的Richard Patten博士，并且在Huang等人的出版物中被修改和利用。 （2012）。