Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations.
Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.
Jessica S
JS
Sadick
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.|Center for Biomedical Engineering, Brown University, Providence, RI, USA.
Molly E
ME
Boutin
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.|Center for Biomedical Engineering, Brown University, Providence, RI, USA.|Brown Institute for Brain Science, Brown University, Providence, RI, USA.|School of Engineering, Brown University, Providence, RI, USA.
Diane
D
Hoffman-Kim
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.|Center for Biomedical Engineering, Brown University, Providence, RI, USA.|School of Engineering, Brown University, Providence, RI, USA.|Department of Orthopaedics, Brown University, Providence, RI, USA.
Eric M
EM
Darling
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