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).

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 <ol><li>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，用记号笔。 </li></ol> 2，解剖相应的脑区<ol><li>颈椎脱位或断头牺牲动物。众议院和安乐死的动物，根据动物保健机构和政府的政策。 </li><li>快速去除日ê大脑从冷冻解剖块头骨和地点。 </li><li>从新鲜组织解剖相应的大脑区域，并继续执行步骤3。 </li></ol> 3，组织匀浆用电机驱动玻璃特氟隆均质机<ol><li>将解剖组织50-100毫克（为SPM）或100-200毫克（为PSD），在含有4毫升的0.32M HEPES缓冲蔗糖溶液标记13毫升聚丙烯管。将样品转移到15毫升锥形玻璃铁氟龙组织研磨机/均质器，设置电机驱动器900转（设置7），均质12招在30秒时段的样本。使用不同的玻璃匀浆器特氟龙样品之间，或用清水冲洗样本之间的冷蒸馏水的均质机，用的Kimwipe擦干。注:最多4个克组织的可匀浆于4毫升的0.32M HEPES缓冲蔗糖溶液，但最好是使用较少的组织，以确保适当的分级分离。 </li><li>均匀化的样本传送回吨Ø同13毫升聚丙烯管。储备100微升匀浆并存储在-80℃用于随后的蛋白质定量和总蛋白级分的免疫印迹分析。 </li></ol> 4，低速离心去除细胞核部分（收益率上清S1） <ol><li>离心机在4℃下将匀浆物在一个固定角转子，在900×g离心10分钟。上清液（S1）转移到一个新的，标为13毫升管和悬浮细胞核部分沉淀（P1），在500微升的0.32M HEPES缓冲蔗糖。注:（P1）的部分可被存储在-80℃下，并用于核级分的免疫印迹分析。 </li></ol> 5，丰富的粗突触体部分的（球团产量P2） <ol><li>离心上清液（S1）以10,000 xg离心15分钟，在4℃下。取出上清液（S2）和储备500μL的微量离心管中保存在-80℃，随后蛋白定量ification和胞浆/轻膜部分免疫印迹分析。重悬剩余的粗突触体部分沉淀（P2）在1ml的0.32M HEPES缓冲蔗糖溶液，然后添加另一毫升0.32的3M HEPES缓冲蔗糖溶液。 </li><li>离心机以10,000×g进行15分钟，在4℃下。取出上清液（S2&#39;），并储备500μL的微量离心管中保存在-80℃，随后蛋白定量和胞浆/轻膜部分免疫印迹分析。保存洗涤的粗突触体丸粒（P2&#39;）的聚丙烯管中。 </li></ol>在原油突触体部分的6裂解（低渗休克） <ol><li>裂解的粗突触体丸粒（P2&#39;）的聚丙烯管中再悬浮是在1ml的DDH 2 O的添加另一个3毫升DDH 2 O的，转移到玻璃铁氟龙组织匀浆，并经均质手3招。注意:这是很重要的以尽可能快地执行此步骤，并迅速进行到步骤6.2。 </li><li>从均化器放回同一13毫升聚丙烯管迅速转移样品调整样本回至4毫米的HEPES和16微升的1M HEPES溶液，颠倒混合。 </li><li>转动样品，在4℃下进行30分钟以确保完全裂解。 </li></ol> 7，丰富的突触膜部分（P3） <ol><li>离心机以25,000×g离心20分钟，在4℃下。除去粗泡状部分上清液（S3），并保留其在5毫升管存放于-80℃，随后蛋白定量和Western blot分析。重悬的突触体膜部分（P3）在1ml的0.32M HEPES缓冲蔗糖溶液。 </li></ol> 8，制备连续蔗糖梯度<ol><li>吸管准确3.5毫升的1.2M HEPES缓冲蔗糖溶液和准确3.0毫升每1.0摩尔和0.8摩尔HEPES-缓冲蔗糖溶液为单独6毫升卡口盖管。注意:这是为了预先测量，用于使蔗糖梯度的缓冲液的体积。卷的精确测量是非常重要的，以确保所得到的梯度将在超速离心平衡。 </li><li>使用玻璃巴斯德吸管和灯泡，转移的1.2M HEPES缓冲蔗糖溶液的12毫升异质同超速离心管。小心层上的1.2M HEPES缓冲蔗糖溶液的顶部的1.0M HEPES缓冲蔗糖溶液。最后，层上的1.0M HEPES缓冲蔗糖溶液的顶部的0.8M的HEPES缓冲蔗糖溶液。使用新鲜的巴斯德吸管每个蔗糖溶液，并注意不要打扰蔗糖梯度。从板凳漩涡或离心振动会干扰梯度的完整性。 </li><li>使用巴斯德吸管，层上的准备连续蔗糖梯度顶部的悬浮突触膜部分（P3）。确保该梯度是通过称量它们的水平​​转头的水桶内平衡。如果水桶是不均衡的，使用P200移液管小心地添加的0.32M HEPES缓冲蔗糖梯度的顶部和平衡转子轮叶。 </li></ol> 9分馏的突触质膜（SPM） <ol><li>超速离心机的水平转头150000 XG 2小时，4℃。小心地从超速离心桶取出蔗糖梯度。用18G的针和1ml注射器，穿刺管在1.0米/ 1.2M的HEPES缓冲蔗糖溶液相间的底部，并撤回突触质膜层（SPM）。 </li><li>记每个SPM样品占据的1ml注射器中的体积。将收集到的SPM层在3.5mL厚壁超速离心管中，添加恰好2.5体积的4 mM的HEPES调节从1.2M的蔗糖浓度为0.32 M。一旦各样品已经调整编到0.32M蔗糖，平衡管和0.32M的HEPES缓冲蔗糖溶液。 </li><li>超速离心机在一个固定角转头200000×g离心30分钟，在4℃。拆下并丢弃上清液。悬浮突触质膜颗粒（SPM）的300微升50毫米肝素钠/ 2毫米的EDTA溶液。直到用于免疫印迹商店的样品在-80℃。 </li></ol> 10，准备突触后密度的分数（PSD） <ol><li>解冻的SPM样品在冰上。 </li><li>让0.54％的Triton X-100的50毫米肝素钠/ 2毫米EDTA溶液的解决方案。 </li><li>在5ml的聚苯乙烯管中，结合2.7的Triton X-100 / HEPES / EDTA溶液和300μl的SPM分数毫升，旋转样品15分钟，在4℃下</li><li>转移样品至3.5毫升厚壁超速离心管并离心样品在32,000 xg离心20分钟，在4℃下。 </li><li>保留上清液（曲拉通X-100可溶组分[TS]）​​，并储存在-80;°℃。注:在此阶段，将沉淀表示"PSD-1T"分数。用洗衣粉第二次治疗将产生一个"PSD-2T"部分。如果PSD-1T部分需要，继续执行步骤10.6。 <ol><li>以产生一个PSD-2T馏分，重悬在3ml 0.5％的Triton粒料X-100的50mM HEPES / 2mM EDTA的溶液中。旋转样品15分钟，在4℃下。以32,000×g离心20分钟，在4℃下离心样品，并继续执行步骤10.6。 </li></ol></li><li>弃上清，重悬突触后（PSD）颗粒在50毫米肝素钠/ 2 mM的EDTA溶液。注:该悬浮量可取决于颗粒的大小;建议50-75微升的体积。由于PSD的分数是洗涤剂不溶部分，通常需要加入1-2微升0.5％SDS中以使蛋白质的完整的再悬浮。直到用于免疫印迹商店的样品在-80℃。 </li></ol>

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The authors have no conflicts of interest to disclose.

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;如果该蔗糖溶液的摩尔浓度是不正确的程序将不工作。因此，蔗糖溶液应当由称量蔗糖，溶解在4毫米的HEPES溶液，然后加入4毫米的HEPES的精确的最终体积来制备。故障排除技巧:建立与准备好的蔗糖溶液测试梯度EN确保该渐变被正确组装。添加不同浓度的溴酚蓝的两个三蔗糖溶液，以协助在三个不同的层中的可视化（例如，最后的0.005％-0.02％重量/体积的浓度）。 重要的是要限制的时间量时的梯度被组装时的离心试管之间。正常扩散会破坏随时间的梯度，并且如果有振动的地方的梯度被存储在工作台上这个被加速。预吹打解决方案集成到单个等分允许精确的量能与巴斯德玻璃吸管吸取。这也降低梯度组件和超速离心的时间。故障排除技巧:打造为同时为蔗糖梯度的实验上述"测试梯度"与溴酚蓝染料。渐变的完整性从而可以随时间监测。测试梯度将作为扩散的指示器，如果梯度组装太远提前或暴露于振动。 最后，收集在尽可能小的体积尽可能的SPM样品，这是因为需要给随后稀释样品用2.5倍体积的水是很重要的。理想情况下，样本应收集在用1毫升注射器0.4〜0.7毫升的体积。明智的做法是在最后20分钟旋在步骤7中建立不连续梯度限制坡度组装和超速的时间。 该协议包括预留所有不同的馏分，并在-80℃下用于随后的分析中存储这些建议。我们已经证明，在图2中突触后蛋白的富集只有这些分数。然而，最好是测量蛋白质的感兴趣的层面中各组分的理解及其分布。比较DISTRIB与其他亚细胞标记蛋白的级分的蛋白质的ution也将有助于确认该实验条件下都达到了预期的分馏。 表2总结了常用的蛋白质标记物，可以用来作为指标进行分馏的阶段8,27- 38。 的不连续蔗糖梯度的利用率，广泛用于富集的突触质膜。该方法具有以下优点:通过连续的Percoll梯度中的不连续蔗糖梯度更容易使不要求专门的设备。然而，这种方法可能不足以精确地定位感兴趣的蛋白，而对于这些应用的Percoll密度梯度可能是优选的。

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。

<table><tbody><tr><td>1 M HEPES, pH 7.4</td><td>BioShop</td><td>HEP003.100</td><td></td></tr><tr><td>HEPES</td><td>BioShop</td><td>HEP001.500&amp;nbsp;</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>BioShop</td><td>SUC507.1</td><td></td></tr><tr><td>EDTA</td><td>BioBasic</td><td>EB0185</td><td></td></tr><tr><td>PMSF</td><td>BioShop</td><td>PMS123.5</td><td></td></tr><tr><td>Aprotinin</td><td>BioShop</td><td>APR600.1</td><td></td></tr><tr><td>Leupeptin</td><td>BioShop</td><td>LEU001.1</td><td></td></tr><tr><td>Pepstatin</td><td>BioShop</td><td>PEP605.5</td><td></td></tr><tr><td>Benzamidine</td><td>BioShop</td><td>BEN601.25</td><td></td></tr><tr><td>Sodium fluoride</td><td>BioShop</td><td>SFL001.100</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>BioShop</td><td>SPP310.100</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>BioShop</td><td>SOV664.10</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>BioShop</td><td>GYP001.10</td><td></td></tr><tr><td>Triton X-100</td><td>BioShop</td><td>TRX506.500</td><td></td></tr><tr><td>18 G x 1 &amp;frac12;&amp;rdquo; needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>1 cc syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Glass Teflon homogenizer Kontes Duall 23</td><td>VWR</td><td>KT885450-0023</td><td></td></tr><tr><td>IKA Model RW 16 basic stirrer</td><td>IKA Works</td><td>2572100</td><td></td></tr><tr><td>Sorvall SM-24 fixed angle rotor</td><td>ThermoScientific</td><td>29017</td><td></td></tr><tr><td>Sorvall RC 6 Plus centrifuge</td><td>ThermoScientific</td><td>46910</td><td></td></tr><tr><td>Thinwall polyallomer tubes (13.2 ml)</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>SW 41 Ti rotor swinging bucket</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Beckman L-80 floor ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Thickwall polycarbonate tubes (3.5 ml)</td><td>Beckman Coulter</td><td>349622</td><td></td></tr><tr><td>TLA-100.3 fixed angle rotor</td><td>Beckman Coulter</td><td>349481</td><td></td></tr><tr><td>Beckman TL-100 tabletop ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

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。

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为蔗糖梯度时的蛋白样品添加（黑色箭头）的一个例子。 以下的离心，应该有蛋白级分的清晰条带在每个相间（0.32 / 0.8 0.8 / 1.0 1.0 / 1.2）。除了颗粒应该是在管的底部可见。参见图3C中的例子超速离心后的部分频段。如果馏分仅漫存在于相间，它是可能的梯度被破坏和进一步加工，不建议。 从小鼠纹状体总，SPM和PSD1T蛋白质样品的蛋白质印迹法证明在图2中。突触蛋白等的GluR1，PSD95和CaMKII的的富集，可以通过从总，SPM加载蛋白（10微克）的恒定量的可视化和PSD馏分。 Perisynaptic蛋白质如多巴胺转运蛋白（DAT）富集在SPM的分数，但在随后的洗涤步骤在PSD浓缩过程中被消除。 以确保等效负载，蛋白质水平可通过用丽春红26染色进行可视化的蛋白质印迹膜。基于抗体的"负荷控​​制"往往有文献报道，但是这可能是用词不当，因为突触蛋白可能是差异erentially调节在试验治疗组。如果基于抗体的"负载控制"是需要的，最好是先确定等效蛋白质加载用丽春红染色，然后进行测试几个参考蛋白来识别那些没有在不同实验组变化。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51896/51896fig1highres.jpg" width="500" /> 图1:工作流亚细胞分离，以丰富的SPM和PSD蛋白组分。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51896/51896fig2highres.jpg" width="500" /> 其中，SPM图2代表免疫印迹和PSD1T蛋白的小鼠纹状体10蛋白提取物微克分别在SDS变性解决，10％丙烯酰胺凝胶上，并转移到PVDF膜上。随后这些印迹孵育为αCaMKII，GluR1的，PSD-93和PSD-95的初级抗体，其是突触后密度的所有组件。因此，这些蛋白质被浓缩的SPM和PSD馏分。交替地，将印迹孵育一抗为DAT，它位于在纹状体突触前膜。而DAT富含SPM的分数，它不是在PSD复合物的一部分，和免疫反应中没有PSD分数观察。针对肌动蛋白或钠/钾ATP酶（钠/钾泵）的抗体可以用作装载对照，尽管一些实验条件可能会影响这些蛋白的水平以及因此相应的负载控制，必须根据经验来确定。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51896/51896fig3highres.jpg" width="500" /> 图3。 </st荣&gt; 蔗糖梯度的代表性例子之前和超速离心后。的梯度的制备A.后，应该有三个阶段，当管被举起到的光可见的。白色箭头表示0.8 / 1.0M的蔗糖溶液和1.0 / 1.2M的蔗糖溶液之间的间期。B.当样品在梯度的顶部加载的，它是在0.32M蔗糖溶液中，并搁置在0.8的顶1M蔗糖层。C.离心后，将匀浆物将分离成几个部分。馏分浮在0.8 / 1.0M的蔗糖溶液的相间会富集在髓磷脂膜。以1.0 / 1.2M的蔗糖溶液相间的分数表示所收集用针和注射器的SPM的分数。在管底部的沉淀物富含线粒体蛋白质。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> SOLU系统蒸发散</td><td> HEPES缓冲蔗糖 </td><td> 蛋白酶抑制剂 </td><td> *磷酸酶抑制剂 </td></tr><tr><td>的1M HEPES pH 7.4的</td><td> 0.32M蔗糖在4毫米的HEPES（pH 7.4）中</td><td> 0.25毫米PMSF（股票250毫米，乙醇，1000倍） </td><td> 10毫米氟化钠（股票500毫米，50X） </td></tr><tr><td> 4毫米肝素钠pH值7.4 </td><td> 0.8M的蔗糖在4毫米的HEPES（pH 7.4）中</td><td> 1.5微克/毫升抑肽酶（股票1.5毫克/毫升，1000倍） </td><td> 2.5毫焦磷酸钠（股票250毫米，100倍） </td></tr><tr><td> DDH 2 O </td><td> 1.0M的蔗糖在4毫米的HEPES（pH 7.4）中</td><td> 10微克/毫升亮肽素（股票10毫克/毫升，1000倍） </td><td> 1.0毫米的B-甘油（股票200毫米，200倍） </td></tr><tr><td>的50mM HEPES（pH 7.4）中2mM EDTA的</td><td> 1.2M的蔗糖在4毫米的HEPES（pH 7.4）中</td><td> 0.1毫克/毫升苄脒（股票100毫克/毫升，1000倍） </td><td> 5.0毫钒酸钠（股票500毫米，100倍） </td></tr><tr><td></td><td></td><td> 10微克/毫升胃蛋白酶抑制剂（股票5毫克/毫升乙醇，500X） </td><td></td></tr></tbody></table> 表1所需的试剂 <table border="0" cellpadding="0" cellspacing="0" width="614"><tbody><tr height="21"><td height="21" style="height:21px;width:88px;"> 分数 </td><td style="width:191px;"> 蛋白组分 </td><td style="width:335px;"> 蛋白质标记 </td></tr><tr height="24"><td height="24" style="height:24px;"> P1 </td><td>核</td><td>组蛋白H1的27 </td></tr><tr height="72"><td height="72" style="height:72px;"> S1 </td><td>细胞质/膜</td><td style="width:335px;">钙联接蛋白28（内质网），α-微管蛋白29（骨架），磷酸甘油醛脱氢酶30（细胞溶质），58K高尔基亲TEIN 31（高尔基） </td></tr><tr height="24"><td height="24" style="height:24px;"> P2和P2&#39; </td><td>原油突触</td><td style="width:335px;"> AMPA受体和NMDA受体亚基32 </td></tr><tr height="48"><td height="48" style="height:48px;"> S2和S2&#39;中</td><td>细胞质/膜的光</td><td style="width:335px;"> nNOS1 29（细胞质），磷酸甘油醛脱氢酶（细胞质），LAMP1 33（溶酶体），PEX14 34（过氧化物酶体） </td></tr><tr height="45"><td height="45" style="height:45px;"> P3 </td><td>突触/线粒体</td><td style="width:335px;"> VDAC 35（线粒体），AMPA受体和NMDA受体亚基（突触） </td></tr><tr height="24"><td height="24" style="height:24px;"> S3 </td><td>突触小泡</td><td style="width:335px;"> SV2 8，突触8 </td></tr><tr height="24"><td height="24" style="height:24px;"> 0.8米/ 1.0M的</td><td>米<html>椰林</td><td style="width:335px;">髓鞘碱性蛋白36 </td></tr><tr height="45"><td height="45" style="height:45px;"> 1.0米/ 1.2M的</td><td> SPM </td><td style="width:335px;"> AMPA和NMDA受体亚基，突触标记（SNAP25 8），neurexin 32的neuroligin 32 </td></tr><tr height="24"><td height="24" style="height:24px;"> 120万像素</td><td>线粒体</td><td style="width:335px;"> VDAC 35 </td></tr><tr height="61"><td height="61" style="height:61px;"> PSD（1T） </td><td> PSD </td><td style="width:335px;"> PSD93 32，PSD95 32，AMPA受体和NMDA受体亚单位的neuroligin，PICK1 32，32的CaMKII </td></tr><tr height="24"><td height="24" style="height:24px;"> TS </td><td>突触前膜</td><td style="width:335px;"> SNAP25，Munc18 37，巴松管38，neurexin </td></tr><tr height="43">飞行:43px;"&gt; PSD-2T </td><td>丰富的PSD </td><td style="width:335px;"> PSD95，AMPA受体和NMDA受体亚基，PICK1，CaMKII的</td></tr></tbody></table> 表2的蛋白标记列表，以识别亚细胞组分

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

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。

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Nanyang Technological University

Research

JoVE Journal

Neuroscience

31.1K 次观看.  University of Toronto. 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。 

视频: Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

科研

生物化学

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

神经科学

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

密度梯度超速离心研究哺乳动物细胞内吞循环

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

分离线粒体接触位点

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

作者聚焦:揭开线粒体接触位点和架构见解 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

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脑膜分离法

核糖体剖面对芽殖酵母中翻译的全基因组定量

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

密度梯度超速离心研究哺乳动物细胞内吞循环

无需梯度测定器或分馏系统的多聚体分析

一种分离线粒体接触位点的改进方法

一种分离线粒体接触位点的改进方法

一种分离线粒体接触位点的改进方法

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后