线粒体相关的ER膜（MAMS）和鞘糖脂丰富的微区（GEMS）：分离小鼠脑
Summary
此过程说明了如何分离从成年小鼠的大脑相关的线粒体的ER膜或MAMS和糖脂富集的微区部分从MAMS和线粒体的准备工作。
Abstract
细胞内细胞器是高度动态的结构具有不同的形状和组合物，该组合物进行特定小区内在的和外在的线索。他们的膜通常定义的接触点，成为中心的交流信号分子和膜元件1,2,3,4并列。间细胞器的膜微区之间形成的内质网（ER）和线粒体在开幕式的IP3敏感的Ca 2 +通道被称为线粒体相关联的的ER膜或MAMS 4,5,6。蛋白质/脂质组成，这些膜接触位点的生化特性已被广泛研究，特别是在关系到他们的作用，调节细胞内Ca 2 + 4,5,6。的ER作为主存储区中细胞内Ca 2 +，和在这容量调节下游无数细胞过程的Ca 2 +信号，包括UDING翻译后的蛋白质折叠和蛋白maturation7。线粒体中，在另一方面，保持Ca 2 +的稳态，通过缓冲胞质Ca 2 +浓度，从而防止细胞凋亡途径的起始下游的Ca 2 +的不平衡4,8。的MAMS的动态特性使得他们的理想地点解剖的基本的蜂窝机制，包括Ca 2 +信号和调节线粒体Ca 2 +浓度，脂质的合成和运输，能量代谢和细胞的存活4,9,10,11,12。已经描述了几种协议用 于纯化这些微区的从肝组织和培养的细胞13,14。
考虑到先前公布的方法，我们已经适应了从成年小鼠的大脑中的线粒体和MAMS隔离的协议。在这个过程中，我们增加了一个额外的纯化步骤，即的Triton X100抽取的，其中Enables糖脂丰富的微区的部分（“创业板”）的MAMS的隔离。这些创业板制剂共享几个蛋白质组分与小窝和脂筏，来自质膜或其他细胞内的膜，以及，提出充当收集聚类受体蛋白质点和用于蛋白质-蛋白质相互作用4,15。
Protocol
该协议的目的是MAMS和宝石鼠脑的分离和纯化解决方案需要分离线粒体，MAMS和宝石的分馏，得到粗线粒体准备： 溶液A：0.32 M蔗糖，1毫摩尔的NaHCO 3，1毫摩尔的MgCl 2，0.5mM的CaCl 2的 +蛋白酶抑制剂（添加新鲜的，根据需要） 溶液B：0.32 M蔗糖，1毫摩尔的NaHCO 3<…
Representative Results
根据我们的经验，使用这个协议，我们可以放心地推荐它从小鼠脑的MAMS，宝石和线粒体组分的分离和纯化。所概述的过程是高度重复性和一致性。在图1中，我们显示的途纯的线粒体和MAMS层上的Percoll梯度（3.4）的代表图像。纯化阿义，含乳白色频带的线粒体（三刀-P），在超离心管的底部偏析，当MAM馏分拼成一个以上的线粒体的漫反射和宽频带。仔细回收个别梯度带后，再悬浮在纯?…
Discussion
细胞内的膜或细胞器和细胞的质膜之间的站点之间的接触代表动态信令平台的基本的细胞过程。他们的生理和病理条件下的功能和组成的准确表征需要可靠和可重复的纯化方法。 MAMS和相应的宝石从成年小鼠的大脑的分离和纯化了专门的优化，通过我们的实验室方法详见这里。该协议已经成功实现了识别指定这些微区的分子效应，和背后的凋亡通路下游的Ca 2 +失衡导致神经细胞死亡的一种…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们做出的贡献的雷纳塔佐野在构思的初步协议。 Ad'A。主席赋予遗传学和基因治疗的儿童（JFC）持有的珠宝商。这部分工作是由美国国立卫生研究院拨款GM60905，DK52025及CA021764，和美国的黎巴嫩叙利亚相关慈善（ALSAC）。
Materials
| Name of Reagent/Material | Company | Catalogue Number | Comments |
| REAGENTS | |||
| Fractionation | |||
| Sucrose | Fisher Scientific | S5-500 | |
| Sodium Bicarbonate | Sigma-Aldrich | S-5761 | |
| Magnesium Chloride, Hexahydrate | Fisher Scientific | BP214-500 | |
| Calcium Chloride, Dihydrate | Sigma-Aldrich | C-5080 | |
| MAM | |||
| D-Mannitol | Sigma-Aldrich | M9546-250G | |
| Hepes | Fisher Scientific | BP310-500 | |
| EGTA | Sigma-Aldrich | E4378-250G | |
| BSA, Fraction V, Heat Shock, Lyophilizate | Roche | 03-116-964-001 | |
| Percoll | GE | 17-0891-02 | |
| GEM | |||
| Triton X-100 | Sigma-Aldrich | T9284-500 ml | |
| Sodium Chloride | Fisher Scientific | S271-3 | |
| Tris Base | Roche | 03-118-142-001 | |
| HCl | Fisher Scientific | A144S-500 | |
| EDTA | Fisher Scientific | BP120-500 | |
| Sodium Dodecyl Sulfate (SDS) | Fisher Scientific | BP166-500 | |
| Common | |||
| Protease Inhibitors Tablets, Complete EDTA-free | Roche | 11-873-580-001 | |
| EQUIPMENT | |||
| 2 ml Douce All-Glass Tissue Grinders | Kimble Chase | 885300-0002 | |
| 15 ml Polypropylene Conical Centrifuge Tubes, BD Falcon | BD | 352097 | |
| 30 ml Round-Bottom Glass Centrifuge Tubes | Kimble Chase | 45500-30 | |
| 15 ml Round-Bottom Glass Centrifuge Tubes | Kimble Chase | 45500-15 | |
| Ultracentrifuge tubes, Ultra-Clear, Thinwall, 14×89 mm | Beckman-Coulter | 344059 | |
| Parafilm | Cole-Parmer | PM996 | |
| Disposable Borosilicate Glass Pasteur Pipets, 9″ | Fisher Scientific | 13-678-20C |
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Citar este artigo
Annunziata, I., Patterson, A., d’Azzo, A. Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain. J. Vis. Exp. (73), e50215, doi:10.3791/50215 (2013).