Small-scale isolation of synaptic vesicles from mammalian brain
Small-scale isolation of synaptic vesicles from mammalian brain
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DOI：
10.1038/nprot.2013.053
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发表时间：
2013-05-01
期刊：
影响因子：
14.8
通讯作者：
Jahn, Reinhard
中科院分区：
文献类型：
--
作者：
Ahmed, Saheeb;Holt, Matthew;Jahn, Reinhard
突触囊泡（SV）是参与神经元释放神经递质的重要细胞器。纯化的SV的生化分析有助于鉴定参与胞吐膜融合和神经递质摄取的蛋白质。尽管已经发表了许多详细描述从大脑中分离SV的方案，但那些提供最高纯度囊泡的方案通常产量较低。在这里，我们描述了一个协议的小规模分离SV从小鼠和大鼠的大脑。该程序依赖于标准的分馏技术，包括差速离心，速率区带离心和尺寸排阻色谱，但它已被优化为最小的囊泡损失，同时保持高纯度。该方案可以在不到1天内完成，并允许从单个小鼠脑中回收类似于150 μ g的囊泡蛋白，从而允许从转基因小鼠中分离囊泡。
Synaptic vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of similar to 150 mu g of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice.