Quantitative Detection of Nucleocytoplasmic Transport of Native Proteins in Single Cells.

Abstract

The detection of protein translocation (i.e., the movement of intracellular proteins among various subcellular compartments) conventionally relies on imaging and subcellular-fractionation-based techniques that do not generate information on a large cell population with single-cell resolution. Although special flow cytometric tools such as imaging flow cytometry may generate single-cell data on processes such as nucleocytoplasmic transport, such equipment is expensive (thus has limited accessibility) and has low throughput for examining cells due to the reliance on high-speed imaging. Here we describe a protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. We conduct chemical release of cytosolic proteins and fluorescence immunostaining of a targeted protein. The detected fluorescence intensity is quantitatively correlated to the cytosolic/nuclear localization of the protein at the single cell level. Our technique provides a simple route for studying nucleocytoplasmic transport with single-cell resolution using common flow cytometers.