准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes
Summary
一个准备从小鼠大脑皮质翻译活跃，完好synaptoneurosomes（SNS）的方法来描述。该方法采用一个不连续Percoll蔗糖密度梯度，允许快速制备活跃的SNS。
Abstract
Synaptoneurosomes（SNS）小鼠大脑皮质的同质化和分馏后获得的。他们再封好，水疱或离开时，皮层组织匀浆脱离的轴突终端的隔离终端。 SNS的保留前和突触后的特点，这使得它们在突触传递的研究非常有用。他们保留用于神经信号的分子机制，并能够吸收，储存和释放神经递质。
积极SNS的生产和隔离可能会出现问题的使用聚蔗糖，可细胞毒性，并要求延长离心由于密度高，过滤和离心的方法，它可以在低活动导致机械损伤的SNS等媒体。然而，使用不连续Percoll蔗糖密度梯度分离SNS提供了一种快速的方法来产生良好收益的翻译活跃的SNS。 Percoll蔗糖梯度法快速，温和的，因为它采用等渗条件下，有越来越短的离心旋转，并避免离心步骤，颗粒SNS和造成机械损伤。
Protocol
1。准备工作1-4 准备一个2.5米的蔗糖浆混合50毫升，500毫升（GM），梯度介质的缓冲，2.5毫升的一个1M的Tris -盐酸，PH7.5股票，0.1毫升0.5 M EDTA，pH值8.0股毫Q水体积。过滤消毒的解决方案，分装和储存冷冻。 准备的河豚毒素（TTX）的1000倍的股票，由1毫米的解决方案中的 Milli – Q H 2 O TTX的等分可以储存在-20 ° C 准备由Milli – Q水解决了100毫米的Tris（pH值7.5），5毫米?…
Discussion
不连续Percoll蔗糖梯度准备此处所描述的是一个快速，可靠的方法来隔离活跃的SNS，它可以用于各种突触传递的实验。此渐变的方法，它是根据Dunkley等人，3,4开发的方法是一种细胞内的脑分馏过程分离前和突触后膜囊泡与另一个相关的衍生。没有进一步纯化这些SNS兼容多种分子生物学技术，包括免疫，免疫印迹，流式细胞仪，监测从头蛋白质的合成，通过[35S] – MET注册成立后，电子?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢来自威斯康星大学麦迪逊分校电子显微镜设施电子显微镜的大学BK八月。这项工作是由NIH资助R01 – DA026067和P30 – HD03352（JSM）的支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Micro BCA Protein Assay Kit | Pierce | 23235 | |
| CaCl2 | Fisher | C79-500 | |
| CO2 gas | Airgas (UW-MDS) | CD 50 | |
| EDTA | RPI | E57020 | |
| EtOH | Fisher | A407SK-4 | |
| HCl | Fisher | A142-212 | |
| Percoll | GE Healthcare | 17-0891-01 | |
| KH2PO4 | Fisher | P285-500 | |
| PierceSDS-PAGE Sample Prep Kit | Pierce | 89888 | |
| NaCl | RPI | S23020 | |
| NaHCO3 | Fisher | BP328-500 | |
| Na2PHO4 | Fisher | S381-500 | |
| Sucrose | RPI | S24060 | |
| Tris Base | RPI | T60040 | |
| Tetrodotoxin | Sigma | T5651 | |
| Express Pro Label Mix S35 Easy Tag | Perkin Elmer | NEG772 | |
| Equipment | Company | Catalogue number | Comments (optional) |
| Dissection tools | |||
| Dounce homogenizer, 7 mL (comes with two glass pestles labled ” A” and “B”) | Wheaton | ||
| P1000 Gilson Pipetman | Gilson | F123602 | |
| Allegra 6KR Centrifuge | Beckman Coulter | 366830 | |
| GH 3.8 Rotor, Swinging bucket rotor | Beckman Coulter | 360581 | |
| Beckman J2-21 Centrifuge | Beckman | ||
| Beckman tubes with caps | Beckman | 355672 | |
| White walled adapters | Beckman | 342327 | |
| Blue walled adapters | Beckman | ||
| JA-17 Rotor, Fixed-angle rotor | Beckman | 369691 |
Table of antibodies used for western blots:
| Name of Antibody | Company | Catalogue number | Host Species | Dilution Factor |
|---|---|---|---|---|
| β-Actin | Sigma | A5441 | mouse | 1:2000 |
| GFAP | Santa Cruz | sc-65343 | mouse | 1:200 |
| GP73 | Santa Cruz | Sc-134509 | rabbit | 1:200 |
| HSC70 | Santa Cruz | sc-7298 | mouse | 1:200 |
| Laminβ | Santa Cruz | Sc-6261 | goat | 1:200 |
| Prohibitin | Santa Cruz | sc-28259 | rabbit | 1:200 |
| PSD95 | Millipore | MAB1596 | mouse | 5 μg/μL |
| SNAP25 | AbCam | ab5666-100 | rabbit | 1:2000 |
| Synaptophysin | Millipore | MAB368 | mouse | 1:500 |
| β-3-Tubulin | Santa Cruz | sc-80016 | mouse | 1:200 |
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Tags
SynaptoneurosomesMouse CortexDiscontinuous Percoll-Sucrose Density GradientHomogenizationFractionationResealed VesiclesIsolated TerminalsSynaptic TransmissionPre- And Postsynaptic CharacteristicsNeuronal SignalingNeurotransmittersFicollCytotoxicCentrifugationFiltrationMechanical DamagePercoll-sucrose Gradient MethodTranslationally Active SNsIsotonic Conditions
Cite This Article
Westmark, P. R., Westmark, C. J., Jeevananthan, A., Malter, J. S. Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient. J. Vis. Exp. (55), e3196, doi:10.3791/3196 (2011).