151. Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
- Author
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Marc López-Cano, Rodrigo A. Cunha, Xavier Morató, Francisco Ciruela, and Paula M. Canas
- Subjects
0301 basic medicine ,General Immunology and Microbiology ,General Chemical Engineering ,General Neuroscience ,030105 genetics & heredity ,Biology ,Neurotransmission ,Protein subcellular localization prediction ,General Biochemistry, Genetics and Molecular Biology ,Transport protein ,Cell biology ,03 medical and health sciences ,Membrane protein ,Postsynaptic potential ,Synaptophysin ,biology.protein ,Phosphorylation ,Function (biology) - Abstract
Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.
- Published
- 2017