title: 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.
authors.affiliation: School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA, 24061, USA.
authors.forename: Zhenning
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Department of Chemical Engineering, Virginia Tech, Suite 235 Goodwin Hall, 635 Prices Fork Road, Blacksburg, VA, 24061, USA. changlu@vt.edu.
authors.forename: Chang
authors.initials: C
authors.lastname: Lu
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism*; D021381:Protein Transport; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D016328:NF-kappa B
keywords: Flow cytometry; Immunostaining; NF-κB; Selective release; Translocation