Abstract
We here describe the methodology to carry out a fractionation procedure that separates presynaptic, postsynaptic, and extrasynaptic fraction, allowing to access the localization of proteins within synapses, which we exemplify in rodent and human brain tissue. The procedure begins with the fractionation of synaptosomes (synaptic compartments) and then combines light solubilization with mild detergents together with alterations of pH to allow a separation by gradient and isopycnic centrifugations of the different subsynaptic compartments. The subsequent use of these fractions in Western blot analysis allows the required validation with markers of each synaptic compartment and enables a direct identification of the localization of any particular synaptic receptor. Although it does not allow the detailed mapping achieved by electron microscopy, the possibility of concentrating the samples allows a greater sensitivity.
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Acknowledgments
We would like to thank Anna Pliássova and Ana Xavier for contributing to the figure and Paula Agostinho, who helped reviewing the protocol. This work was supported by the Defense Advanced Research Projects Agency (DARPA, grants 09-68-ESR-FP-010 and W911NF-10-1- 0059), the Portuguese Foundation for Science and Technology (FCT, PEst-C/SAU/LA0001/2013–2014), and Quadro de Referência Estratégica Nacional (QREN, CENTRO-07-ST24-FEDER-002006).
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Canas, P.M., Cunha, R.A. (2016). Subsynaptic Membrane Fractionation. In: Luján, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 110. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3064-7_3
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DOI: https://doi.org/10.1007/978-1-4939-3064-7_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3063-0
Online ISBN: 978-1-4939-3064-7
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