Receptor and Ion Channel Detection in the Brain - Methods and Protocols
Receptor and Ion Channel Detection in the Brain - Methods and Protocols
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大脑中的受体和离子通道检测 - 方法和方案
DOI：
10.1007/978-1-4939-3064-7_4
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Molnár E
中科院分区：
文献类型：
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作者：
Molnár E
神经递质受体的细胞表面运输和内吞作用是神经传递的重要调节机制。脑切片中质膜蛋白的生物素化允许它们与存在于细胞内细胞器中的那些分离。膜不可渗透、硫醇可裂解和胺反应性生物素化试剂（例如EZ-连接磺基-NHS-SS-生物素）与表面暴露蛋白质的伯氨基形成稳定的共价键。在脑切片的均质化和膜的溶解之后，可以用连接到琼脂糖珠的亲和素或链霉亲和素分离生物素标记的蛋白质。结合的生物素化蛋白质在还原剂（例如谷胱甘肽或β-巯基乙醇）存在下从亲和素或链霉亲和素中释放。生物素标记的（表面）和未标记的（细胞内）蛋白质组分的分子组成的定量差异可以使用免疫印迹与靶蛋白特异性抗体进行分析。虽然这个程序的许多变化存在于文献中，在这一章中，我们描述了生物素化协议，我们已经申请的定量变化的细胞表面表达和内化的离子型谷氨酸受体在急性脑切片的调查。
Cell surface trafficking and endocytosis of neurotransmitter receptors are important regulatory mechanisms of neurotransmission. Biotinylation of plasma membrane proteins in brain slices allows their separation from those present in intracellular organelles. Membrane-impermeable, thiol-cleavable and amine-reactive biotinylation reagents (e.g. EZ-link sulfo-NHS-SS-biotin) form a stable covalent linkage with primary amino groups of surface-exposed proteins. Following homogenisation of brain slices and solubilisation of membranes, biotin-labelled proteins can be isolated with avidin or streptavidin linked to agarose beads. Bound biotinylated proteins are released from avidin or streptavidin in the presence of reducing agents (e.g. glutathione or β-mercaptoethanol). Quantitative differences in the molecular composition of biotin-labelled (surface) and unlabelled (intracellular) protein fractions can be analysed using immunoblotting with target protein-specific antibodies. While many variations of this procedure exist in the literature, in this chapter we describe the biotinylation protocol that we have applied for the investigation of quantitative changes in the cell surface expression and internalisation of ionotropic glutamate receptors in acute brain slices.