Abstract
The blood–brain barrier (BBB) represents 95% of the molecular exchange area between the blood and the cerebral compartment. The BBB plays a major role in the brain homeostasis, essentially by regulating the passage of endogenous and exogenous circulating compounds. There is direct and indirect evidence that proteins such as membrane transporters, enzymes, and tight junction proteins interact to establish and maintain this metabolic and physical cellular barrier. First, we describe a method for harvesting of brain capillary endothelial cells (BCECs) from our in vitro BBB model. Then, we develop the protein preparation (cell lysis and protein isolation) and the two-dimensional gel electrophoresis steps, including the isoelectric focusing and SDS-PAGE conditions, the gel staining, the image acquisition, and the comparative study. Finally, we present examples of protein identification by peptide mass fingerprint (PMF) measured by MALDI-TOF-MS analysis, and complementary data issued from peptide fragmentation fingerprints (PFF) that allow successful protein identifications. PMF and PFF analyses provide complementary datasets and thus, a more comprehensive sequence coverage of the BCEC proteome, especially when they are combined together (PMF/PFF).
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Duban-Deweer, S., Flahaut, C., Karamanos, Y. (2012). The Proteome of Brain Capillary Endothelial Cells: Toward a Molecular Characterization of an In Vitro Blood–Brain Barrier Model. In: Karamanos, Y. (eds) Expression Profiling in Neuroscience. Neuromethods, vol 64. Humana Press. https://doi.org/10.1007/978-1-61779-448-3_10
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DOI: https://doi.org/10.1007/978-1-61779-448-3_10
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