从大鼠坐骨神经轴浆隔离 (Translated to Chinese)

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Cite this Article: 从大鼠坐骨神经轴浆隔离

Rishal, I., Rozenbaum, M., Fainzilber, M. Axoplasm Isolation from Rat Sciatic Nerve. J. Vis. Exp. (43), e2087, doi:10.3791/2087 (2010).

Abstract: 从大鼠坐骨神经轴浆隔离

纯周围神经轴突胞浆（轴浆）的分离是许多生物过程的生化研究的关键。在这篇文章中，我们展示和描述基于以下步骤从成年大鼠坐骨神经神经轴浆隔离协议：（1）清扫的神经分册和结缔组织分离;（2）潜伏期短段神经分册低渗介质释放和裂解髓鞘轴突非结构;（3）提取其余的轴突丰富的材料。该制剂的蛋白质组学和生化特性，已证实为轴突组成部分的高度浓缩。

Protocol: 从大鼠坐骨神经轴浆隔离

此协议允许轴浆隔离与胶质细胞和血管组织污染最小化。该方法的产量约为70-100微克每8-10周龄Wistar大鼠共轴浆蛋白质。

1。解剖坐骨神经神经

安乐死两个大鼠颈椎脱位的CO ₂吸入。触诊缺乏心跳开始清扫前确认死亡。
拭清扫面积70％的乙醇。
切开皮肤，单独的肌肉和隔离坐骨神经使用不破坏血管的剪刀和镊子仔细。解剖坐骨神经两个（每个约1.5厘米）从每个动物和收集500μL的PBS 0.2X +抑制剂在Eppendorf公司所有四个神经，不停地在冰。
细分的神经传输含有至少2毫升的PBS 0.2X +蛋白酶抑制剂的塑料菜。
删除从下双目范围内使用罚款钳神经外膜。独立的分册，非常仔细，直到他们变得浑浊，并开始浮于表面的解决方案。这一步应该是练习，直到它可以快速，准确地执行（不超过10％的神经分钟）。

2。孵育，洗涤和洗脱

传输分离分册到一个新的Eppendorf管中，用500的PBS 0.2X为2小时在室温下孵育μL蛋白酶抑制剂。
1毫升转移分册Eppendorf管Eppendorf管中，并摇晃5分钟后，转移相同的缓冲区清洗至少3次。
要删除多余的液体放入一个新的空Eppendorf管分册中，然后转移与1X PBS 300μL蛋白酶抑制剂在室温为20-30分钟含孵化分册到一个新的Eppendorf管。
离心10分钟，在4万XG ° C。
取上清和测量蛋白质浓度。这是你的轴浆准备。

3。代表性的成果

图1。免疫印迹比较轴浆由手动挤压法在本议定书中所示的轴浆隔离样品中分离。注意白蛋白和GFAP的水平降低，血液中的蛋白质和胶质细胞污染，分别为。相比之下，在此准备丰富性轴索微管蛋白B3，表明轴突蛋白的高层次。

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Male Wistar rats (8-10 weeks old) PBS 0.2X and PBS 1X solutions containing protease inhibitors (Roshe) 2 tablets per 50 ml Eppendorfs for 1.5 ml Sterilized plastic dishes 35 mm Dissecting tools including: scissors and fine forceps Binocular and table light
Antibodies
Mouse anti-GFAP clone G-A-5 was from Sigma (G6171). Rabbit anti-Albumin was from Cedarlane (CLAG5140). Mouse anti-Tubulin β3 and rabbit anti-gERK were from Sigma (T2200 and M5670 respectively).

References: 从大鼠坐骨神经轴浆隔离

Hanz, S., Perlson, E., Willis, D., Zheng, J.Q., Massarwa, R., Huerta, J.J., Koltzenburg, M., Kohler, M., van-Minnen, J., Twiss, J.L., et al. Axoplasmic importins enable retrograde injury signaling in lesioned nerve. Neuron 40, 1095-1104 (2003).
Michaelevski, I., Medzihradszky, K.F., Lynn, A., Burlingame, A.L., and Fainzilber, M. Axonal transport proteomics reveals mobilization of translation machinery to the lesion site in injured sciatic nerve. Mol Cell Proteomics 9(5) : 976-87 (2010).
Rishal, I., Michaelevski, I., Rozenbaum, M., Shinder, V., Medzihradszky, K.F., Burlingame, A.L., and Fainzilber, M. Axoplasm isolation from peripheral nerve. Dev Neurobiol 70, 126-133 (2010).
Twiss, J.L., and Fainzilber, M. Ribosomes in axons--scrounging from the neighbors? Trends Cell Biol 19, 236-243 (2009).

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Date Published	9/24/2010
JoVE Section	Neuroscience
JoVE Issue	43
Comments	0
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DOI	doi:10.3791/2087

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从大鼠坐骨神经轴浆隔离

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