突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备
Summary
本文将详细介绍与突触质膜上超速不连续蔗糖梯度相关蛋白的富集。随后的制备突触后密度蛋白也被描述。蛋白制剂适用于免疫印迹或2D DIGE分析。
Abstract
神经元亚细胞分离技术，允许被贩卖，并从突触蛋白的定量。如在1960年代末期最初所述，突触质膜相关蛋白，可以通过超速离心法在蔗糖密度梯度分离。一旦突触膜分离，大分子复合物被称为突触后密度可以随后，由于其不溶于洗涤剂中分离。用于分离突触细胞膜和突触后密度蛋白的技术基本上保持40年后相同的，并且被广泛应用于目前神经科学研究。本文将详细介绍与突触细胞膜及突触后密度采用不连续蔗糖密度梯度相关的蛋白质的分离。导致蛋白制剂适用于免疫印迹或2D DIGE分析。
Introduction
神经元通过突触进行通信，并且该通信质量是通过在蛋白质在突触的组成改变调节到很大程度。特别是位于突触后密度蛋白通过密切脚手架神经递质受体与它们的信号转导系统1参与神经元沟通。此外，在突触效能的强度持久的变化是由于突触后密度1-6添加或去除受体的控制。因此，突触蛋白的分离和定量是一种必要的和有用的技术，来获得有关该神经元响应的刺激和改变突触效能7的方式。本文介绍了一种常见的技术，通过离心分离从啮齿动物脑组织中突触蛋白在连续蔗糖梯度。突触细胞膜部分可以得到丰富和孤立的基础上其在蔗糖密度，已根据经验确定，以类似于1.2M的蔗糖。
根据不同的生物学问题，亚细胞组分可以由蔗糖或珀可连续或不连续梯度进行分离。连续梯度允许蛋白质成多个组分的分离;这可以是特别有用的，以证明一个给定的级分8中的共定位的蛋白质。然而，连续梯度的制备是更费力的和是不必要的许多应用。不连续的梯度是比较容易制备和可用于蛋白分离成几，通常定义的馏分。这是由增加的摩尔浓度的3蔗糖层的不连续梯度，已被广泛用于分离与突触质膜（SPM）相关的蛋白质。本突触质膜级分可被进一步加工，以突触后密度fractioN（PSD）的洗涤剂不溶部分的去污剂处理和隔离。
当这个过程在1960年的9,10首次被描述，电子显微镜被用来证实细胞器及膜的大致限定突触质膜和突触后密度级分9-14。这些研究证明了包含前和突触后膜和在SPM分数突触小泡;洗涤剂治疗主要是电子致密后，突触后密度为可见的。在该过程中，低渗休克用于从细胞体10夹断一样处理。这个步骤需要的事实，线粒体是渗透冲击更耐和保持不动，并且因此它们在蔗糖梯度（ 图1）的底部沉淀的优点。
用同样的富集技术，对SPM和PSD分数分别为第一生化通过聚丙烯酰胺凝胶电泳和主要蛋白质组分15-17的序列来定义。随后免疫印迹分析已被用于检测和定量突触蛋白的水平，并进一步确定这些级分（ 图2）。我们已经使用该技术在我们的实验室进行量化改变多巴胺转运时Slc6a3基因被复制在小鼠18所发生的突触水平。我们也使用这种技术在NMDA受体缺陷小鼠揭示突触特异性降低的蛋白质，是在DISC1相互作用组19的一部分。
它是由免疫印迹分析了SPM的馏分含有突触小泡膜蛋白，核内体标记物，线粒体蛋白质，膜相关合成酶和信号转导分子，以及突触后密度的组成部分和突触质膜20-23明显。 ËVEN PSD级分可具有污染具有丰富的线粒体蛋白质，它可能有必要进行第二梯度沉降或额外的纯化步骤，以除去它们13。最近，定量质谱法提供了超过100个蛋白质在单独的突触后密度的列表，以及这些部件24,25的相对丰度的指示。
Protocol
以下协议符合动物保护的加拿大议会的指引，并已获得医药动物保健委员会的教授在多伦多大学。 1，准备好所需的试剂中的说明表1 添加蛋白酶和磷酸酶（如果需要的话）的抑制剂，以所有的蔗糖溶液，缓冲液和DDH 2 O的以表1所列的浓度。重要：执行所有的步骤在4℃和预冷所有在实验前的起始试剂和设备。标签所有管道，包括超速离心管，用记?…
Representative Results
在蔗糖密度梯度的制备方法应导致蔗糖（0.8，1.0和1.2M的蔗糖）的3摩尔的解决方案的明确分离。参见图3A为渐变的示例的蛋白质样品加入之前。如果梯度制备太多提前，或者如果它被制备的长凳上表面与来自其它设备的振动，梯度将受到损害，并适当分离，将无法实现。如果三种解决方案的明确分离是不可见的，最好是加入的蛋白样品之前，使一个新的梯度。参见图3B…
Discussion
还有在该过程的几个步骤，这对于一个成功的结果至关重要。在步骤3中，重要的是均质化的一致度，实现对每个样品。当均质化组织与马达驱动的均化器，以恒定速度不仅用于杵的旋转，而且还与笔划的数量。在渗透休克的温育时间应精确，因为在低渗溶液中的扩展同质或孵化将裂解线粒体和污染SPM样品。
另一个关键步骤是在试剂，尤其是蔗糖溶液的制备;如果该蔗糖溶液的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).
Materials
| 1M HEPES, pH 7.4 | BioShop | HEP003.100 | |
| HEPES | BioShop | HEP001.500 | |
| Sucrose | BioShop | SUC507.1 | |
| EDTA | BioBasic | EB0185 | |
| PMSF | BioShop | PMS123.5 | |
| Aprotinin | BioShop | APR600.1 | |
| Leupeptin | BioShop | LEU001.1 | |
| Pepstatin | BioShop | PEP605.5 | |
| Benzamidine | BioShop | BEN601.25 | |
| Sodium fluoride | BioShop | SFL001.100 | |
| Sodium pyrophosphate | BioShop | SPP310.100 | |
| Sodium orthovanadate | BioShop | SOV664.10 | |
| β-glycerophosphate | BioShop | GYP001.10 | |
| Triton X-100 | BioShop | TRX506.500 | |
| 18G x 1 ½” needle | BD | 305196 | |
| 1cc syringe | BD | 309659 | |
| Name of Equipment | Company | Catalog number | |
| Glass Teflon homogenizer Kontes Duall 23 | VWR | KT885450-0023 | |
| IKA Model RW 16 Basic Stirrer | IKA Works | 2572100 | |
| Sorvall SM-24 Fixed Angle Rotor | ThermoScientific | 29017 | |
| Sorvall RC 6 Plus Centrifuge | ThermoScientific | 46910 | |
| Thinwall Polyallomer Tubes (13.2mL) | Beckman Coulter | 331372 | |
| SW 41 Ti Rotor Swinging Bucket | Beckman Coulter | 331362 | |
| Beckman L-80 Floor Ultracentrifuge | Beckman Coulter | ||
| Thickwall Polycarbonate Tubes (3.5mL) | Beckman Coulter | 349622 | |
| TLA-100.3 Fixed Angle Rotor | Beckman Coulter | 349481 | |
| Beckman TL-100 Tabletop Ultracentrifuge | Beckman Coulter |
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Cite This Article
Bermejo, M. K., Milenkovic, M., Salahpour, A., Ramsey, A. J. Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient. J. Vis. Exp. (91), e51896, doi:10.3791/51896 (2014).