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 nucleocy-toplasmic 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 with- out 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.
CITATION STYLE
Cao, Z., & Lu, C. (2015). Quantitative detection of nucleocytoplasmic transport of native proteins in single cells. In Methods in Molecular Biology (Vol. 1346, pp. 239–252). Humana Press Inc. https://doi.org/10.1007/978-1-4939-2987-0_16
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