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.
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This research was supported in part by NSF Grant 0967069.
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Cao, Z., Lu, C. (2015). Quantitative Detection of Nucleocytoplasmic Transport of Native Proteins in Single Cells. In: Singh, A., Chandrasekaran, A. (eds) Single Cell Protein Analysis. Methods in Molecular Biology, vol 1346. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2987-0_16
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DOI: https://doi.org/10.1007/978-1-4939-2987-0_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2986-3
Online ISBN: 978-1-4939-2987-0
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