Abstract
Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).
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Acknowledgments
Heather VanGuilder Starkey for sharing her isolation protocols with us. Nicolas Bazan for sending us the rabbit anti-VGLUT2 antibody (Dcf68) cloned by his laboratory.
This work was supported by the following grants: EY024520, EY021716 to WMF; EY023202 to DRM; NS090117, EY004149, EY000871, and EY021725 (P30 Vision Core Grant) to REA; NS089358 to BRH; EY022071, EY025256, to NAM; and Foundation Fighting Blindness to REA and NAM. Unrestricted grant from Research to Prevent Blindness, Inc.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Hopiavuori, B.R. et al. (2023). Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation. In: Bhattacharya, S.K. (eds) Lipidomics. Methods in Molecular Biology, vol 2625. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2966-6_2
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DOI: https://doi.org/10.1007/978-1-0716-2966-6_2
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