Subcellular Fractionation of Cultured Human Cell Lines

Abstract

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 (1), and was modified and utilized in a publication by Huang et al. (2012).