Morató Xavier, López-Cano Marc, Canas Paula M, Cunha Rodrigo A, Ciruela Francisco
Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat; Institut de Neurociències, Universitat de Barcelona.
Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra.
J Vis Exp. 2017 May 12(123):55661. doi: 10.3791/55661.
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.
评估突触蛋白的组成和功能是神经科学领域的一项重要挑战。然而，评估突触内发生的神经传递并非易事，因为它受到动态蛋白质 - 蛋白质相互作用和磷酸化事件的高度调控。因此，当使用任何方法研究突触传递时，一个主要目标是保留这些短暂的生理修饰。在这里，我们提出了一种脑膜分级分离方案，这是一种用于分离属于不同突触区室的蛋白质的可靠方法。换句话说，该方案描述了一种从突触前、突触后和突触外区室进行蛋白质富集的生化方法。首先，通过不连续蔗糖梯度从包含所有突触区室的神经元中获得突触体或突触终末。值得注意的是，这种初始突触膜制备的质量至关重要。随后，在不同pH条件下使用温和的去污剂进行轻度溶解，从而实现不同突触下区室的分离。这允许通过梯度离心和等密度离心进行分离。最后，通过使用特征明确的突触蛋白标记物（即分别为SNAP - 25、PSD - 95和突触素）进行免疫印迹分析，验证不同突触下区室（即突触前、突触后和突触外膜组分）的蛋白质富集情况，从而能够直接评估任何特定神经元蛋白的突触分布。
