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。

1。准备工作1-4 <ol><li>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。 </li><li>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 </li><li>准备由Milli - Q水解决了100毫米的Tris（pH值7.5），5毫米的Na 2 HPO 4，4毫米的KH 2 PO 4， 碳酸氢钠 3 40毫米，800毫米氯化钠10X刺激缓冲区。股票可以储存于4 ° C。 </li><li>预冷离心机到4 ° C。 </li><li>股票的等渗Percoll（SIP）解决方案，并准备加入Percoll（18毫升）的9卷1体积为2.5米的蔗糖（2毫升）在Milli - Q水拌匀。 </li><li> SIP的款额加入到通用汽车缓冲和混合每个准备3，10，15和23％的梯度层: </li></ol><img alt="表0" src="/files/ftp_upload/3196/3196table0.jpg" /><ol start="7"><li>吹打成贝克曼离心管中倒入2毫升，23，15，10和3％的等渗Percoll解决方案使用一个P1000的吉尔森Pipetman帽梯度层。层与层之间的接口应该清楚没有可辨别的混合层。保存在4 ° C，使用前至少20分钟的梯度。梯度可以存储长达24小时，虽然同一天，建议使用。 [没有经验的实验室人员可能加入蓝色染料isomotic Percoll层界面的可视化解决方案的做法准备梯度。] </li></ol> 2。鼠标清扫1 <ol><li>确保所有的鼠标畜牧业和安乐死的程序是按照美国国立卫生研究院和大学准则执行。安乐死的二氧化碳窒息或批准在动物协议的一种方法的幼崽（P13到P21的年龄）。 </li><li>引脚上的鼠标到夹层板和喷涂的颈部和头部用乙醇的背面。通过脊髓削减颅底一个锋利的剪刀，去除皮肤的头骨顶部，横向切割之间的顶骨和interparietal骨之间的大脑和皮层区域的头骨。然后切基地鼻子沿矢状缝合头骨，小心地取出每个半球侧拉软顶骨，并用抹刀删除皮层。插入大脑小脑以上锅铲，然后舀出皮质，并放置在冰冷的通用缓冲区。立即着手同质化的步骤。不要让皮质坐很长时间在冰冷的缓冲区，因为这将造成不利影响形成的SNS。 </li></ol> 3。制备匀浆和SNS 1-4 <ol><li>在冰冷的通用缓冲冲洗皮质和两个皮层转移到玻璃Dounce匀浆含有5毫升冷通用缓冲区。 </li><li>轻轻同质化的松散杵5-10招皮层（杵的"A"），其次是5-10招紧杵（杵的"B"）。招应迅速，但足够慢，不引起气泡。一个匀浆同质化后的图片是在计划 1 。笔画数会有所不同取决于个人匀浆和随行杵。当完成的梯度应给人以强烈的的SN频段，如果不是，调整笔画数。 </li><li>匀浆转移到15 mL锥形1000xg离心10分钟，在4 °在水平转头C（Allegra的6KR离心机）颗粒细胞碎片和核。纺匀浆的照片是在计划 1 。 </li><li>第2层毫升上清液，每Percoll蔗糖梯度，第管，并在32,500 XG离心5分钟，4 °彗星在一个固定角度转子（美国贝克曼离心机J2 - 21，JA - 17转子）使用适当的适配器（ 试剂和仪器表 ）。 </li><li>移液器起飞和丢弃以上SN频段使用玻璃巴斯德吸管的解决方案。移液器关闭SN频段在15/23％接口，转移到一个圆锥形的冰商店。每个梯度之一皮质会产生0.9-1.1毫升锡。 </li><li>调整的SNS盐的浓度，加入十分之一量的10倍刺激缓冲区，增加1000倍氯化钙 2至终浓度为12纳米，1毫米TTX的股票添加终浓度为1μm，抑制非特异性激发。 （ 氯化钙除了是可选的，如果它会干扰与下游SN应用） 。 </li><li>使用的Micro BCA蛋白检测试剂盒，确定了SNS的蛋白浓度。 （Bradford蛋白测定是不建议，因为从Percoll推断。） </li><li> SNS可以用来直接和迅速的蛋白质translaTION研究。对于其他应用程序的SN裂解可能被清理或集中使用皮尔斯的SDS - PAGE样品制备试剂盒。 </li></ol> 4。代表性的成果: 当鼠标皮质匀浆，并在不连续Percoll蔗糖梯度（ 图1），6条带或分数分离被发现（ 图 1 ）。这是以前对大鼠大脑皮层3,5所示的分子量高频段的成分被破坏细胞膜和髓磷脂和颗粒材料中所含的线粒体或细胞器（ 见表1） 。 23％/ 15％的接口（乐队5）含有丰富的SNS。 如前所述，通过电子显微镜（EM）的乐队在23％/ 15％接口包含完整的SNS，删除和检查。2,6电磁显示存在完整的突触小泡与突触前和突触后成分的保存 （图2）。前和突触后车厢分离是很重要的研究信令和蛋白质的合成，其中发生在两个车厢。7-13 Percoll蔗糖梯度是一个粗略的净化，使我们看到在SN分数轻微的细胞，核和细胞器的污染，但整体分离是好的。 用Western blot检测时，Percoll分数包含在SN带（ 图3，表2）增加突触纯度与预期的蛋白标志物。丰富的SN频段（第五级）均保持在突触和神经元的标记，但其他标志物的丰度显着降低。特别是，GFAP的胶质细胞来源的星形胶质细胞和肿瘤细胞的标记，核纤层蛋白B1，核标记，存在几乎可以忽略不计，而维持或加强的结构和突触标记。此外，还有被保留，在蛋白质合成中发挥的作用的细胞器，如线粒体和高尔基的标记。 典型的产量分别为SN频段250-500纳克每微升的蛋白质，BCA蛋白检测确定。 从头蛋白质合成量的确定活动的SNS，目前由[35S]蛋氨酸掺入（ 图 4）作为监测 。2基底翻译活动增加了1.56倍时，SNS与50微米glutamate/10 μM甘氨酸刺激（GLU）或5.12倍的PIN1抑制剂胡桃启动时。 PIN1的抑制作用，增加蛋白质合成。结果证实，[35S]蛋氨酸掺入的增加是由于从头蛋白质的合成，我们增加了40微米茴香霉素，蛋白质合成抑制剂，并看到了在法团的显着下降在基础水平相比，谷氨酸的存在和缺乏。此外，基底翻译时增加2％TRITON - X 100，这破坏了诚信的SNS，下降了75％。此外，当研究表明最突触标记富集乐队（B4 - B6）， SNS或乐队5 从头蛋白质的合成（ 图5）最大的活动。因此，蔗糖不连续Percoll梯度迅速产生一种非常活跃，丰富SN频段，可用于研究蛋白质翻译。 不连续Percoll蔗糖梯度程序比其他方法更有利，因为苛刻的条件所造成的机械损伤。相比的过滤方法，在清除皮质匀浆，是通过一系列过滤器的大小减少，3,14-15 Percoll方法形成了更大的活动更多的SNS。在图6中可以看出，当超过Percoll蔗糖梯度，有远不如SNS在15/23％接口和更大量的碎膜过滤法编制的SNS等分通过。当从头蛋白质合成是通过S35 -会见纳入计量，过滤法编写的SNS远不如积极（ 图7）。为了最大限度地提高翻译活动，我们利用小鼠P14和P18之间。 SN可以从成年小鼠的准备，但我们观察到的显着增加与少年小鼠（ 图8）编写的SN翻译活动。 <img alt="表1" src="/files/ftp_upload/3196/3196table1.jpg" /> 表1。 3,5 匀浆鼠标皮质不连续Percoll梯度分馏带的成分 。 <img alt="表2" src="/files/ftp_upload/3196/3196table2.jpg" /> 表2。每个乐队的分数相对西方螺栓不连续Percoll蔗糖梯度带成分分析总结。蛋白浓度隔离使用不连续的PercolWestern blot法测定L -蔗糖梯度。无蛋白（ - ）目前，0≥0.2（+），0.2≥0.8 (++),或&gt; 0.8 (+++)倍大于蛋白质上清（皮质匀浆，离心），代表目前在N = 3。 <img alt="计划1" src="/files/ftp_upload/3196/3196scheme1.jpg" /> 计划1:一个SN准备使用一个不连续Percoll蔗糖密度梯度的原理图，解剖小鼠大脑皮质，匀浆，并在低速离心除去非均质组织，细胞，整个细胞器， 如原子核和膜。不连续梯度应当有明确定义的层。插图显示的图片，仅供都被染成了蓝色，只有在为了形象化层的层，例如。上清分层不连续Percoll蔗糖梯度上，在中等速度离心。从渐变的SN频段，然后检索，并准备使用。 <img alt="图1" src="/files/ftp_upload/3196/3196fig1.jpg" /> 图1。匀浆分馏上的不连续Percoll蔗糖梯度 。鼠标皮质匀浆，上升至6阶或分数上不连续Percoll蔗糖梯度分离。纯化的，积极的SNS（*）被发现在第五级。 <img alt="图2" src="/files/ftp_upload/3196/3196fig2.jpg" /> 图2。 SN的筹备工作给予了SNS的异构混合保留突触前和突触后成分 。SN的准备从C57BL / 6小鼠（P13到P21的年龄）准备了一份电子显微镜执行先前所描述的，表明SNS保留突触前和突触后的元素。 6个红色箭头指向性突触后密度。比例尺为1微米。 <img alt="图3" src="/files/ftp_upload/3196/3196fig3.jpg" /> 图3。的SN制剂的免疫印迹分析表明，突触和神经元的标记保持在纯化的SNS（第五级），但其他标志物的丰度显着降低。上清免疫印迹的（S，离心皮质匀浆）和不连续频段1-6 Percoll蔗糖梯度被探测的肌动蛋白（β-的结构，装载控制），GFAP（星形），GP73（高尔基），HSC70（细胞质和核），核纤层蛋白B1（核），Prohibitin（线粒体），PSD95（突触后密度），SNAP25（突触），突触（突触）和β3-微管蛋白（神经元特异性）。带可视化使用风暴860 Phosphorimager，N = 3的代表。 <img alt="图4" src="/files/ftp_upload/3196/3196fig4.jpg" /> 图4。 SNS是活动的，展览通过[35S] -蛋氨酸掺入从头蛋白质的合成。SNS预平衡至37 ° C为10分钟。 SNS是未经处理或预处理后15分钟与40微米茴香霉素或1μM胡桃在37 ° C，[35S]此外，气象，20μL快临标签混合S35简易标签，加上或减去谷氨酸37 ° C 30分钟。使用皮尔斯的SDS - PAGE样品制备试剂盒的样品制备，15％的聚丙烯酰胺凝胶上运行，干燥和定量使用一个分子动力学风暴860 phosphorimager，N = 3的代表，± SEM。 <img alt="图5" src="/files/ftp_upload/3196/3196fig5.jpg" /> 图5。从非连续Percoll蔗糖梯度带5条所载从头蛋白质合成的最高水平。乐队4，5和第6和上清液（离心皮质匀浆）预平衡至37 ° C下10分钟。然后，[35S] -蛋氨酸，快临标签混合S35简易标签，增加了，再加上或减去谷氨酸在37 ° C为30分钟。使用皮尔斯的SDS - PAGE样品制备试剂盒的样品制备，15％的聚丙烯酰胺凝胶上运行，干燥和定量使用一个分子动力学风暴860 phosphorimager，N = 3的代表，± SEM。 <img alt="图6" src="/files/ftp_upload/3196/3196fig6.jpg" /> 图6。从过滤法准备的SNS包含更多的碎膜比蔗糖不连续Percoll梯度法准备的SNS少整个SNS。 SNS准备通过一系列如前所述随着孔径的过滤器匀浆皮质 。3,14-15的SNS等分是在一个不连续Percoll蔗糖梯度离心和过滤法编制相比同等数额使用蔗糖不连续Percoll梯度法的材料。 <img alt="图7" src="/files/ftp_upload/3196/3196fig7.jpg" /> 图7。从过滤法编写的SNS包含少从头蛋白质合成活性比SNS准备来回米的不连续Percoll蔗糖梯度法。 SNS准备通过一系列降低孔径的过滤器，如前面所述 ，3,14-15匀浆皮质不连续Percoll蔗糖梯度方法。 SNS都准备工作，预平衡至37 ° C下10分钟。然后，[35S] -蛋氨酸，快临标签混合S35简易标签，增加了，再加上或减去谷氨酸在37 ° C为30分钟。使用皮尔斯的SDS - PAGE样品制备试剂盒的样品制备，15％的聚丙烯酰胺凝胶上运行，干燥和定量使用一个分子动力学风暴860 phosphorimager，N = 3的代表，± SEM。 <img alt="图8" src="/files/ftp_upload/3196/3196fig8.jpg" /> 图8。从年轻小鼠（P13）的SNS有更大的比年纪较大的老鼠（P85）翻译活动 。SNS从不同年龄的小鼠使用Percoll不连续蔗糖梯度法，在37 preequilibrated ° C 10分钟准备，处理或与谷氨酸治疗中存在[35S] -蛋氨酸（快递PRO标签混合S35简易标签）为30分钟，扣冻结，后清理与皮尔斯的SDS - PAGE样品制备试剂盒。 SNS的12％SDS聚丙烯酰胺凝胶上，然后运行，干和分子动力学风暴860 phosphorimager，N = 3，± SEM代表成像。 P14小鼠SNS至少3倍以上的P85小鼠积极。

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没有利益冲突的声明。

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?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>微BCA蛋白检测试剂盒</td><td>刺穿</td><td> 23235 </td><td></td></tr><tr><td> 氯化钙 </td><td>费舍尔</td><td> C79 - 500 </td><td></td></tr><tr><td> CO 2气体</td><td>公司Airgas（UW - MDS） </td><td>光盘50 </td><td></td></tr><tr><td> EDTA的</td><td>零售物价指数</td><td> E57020 </td><td></td></tr><tr><td>乙醇</td><td>费舍尔</td><td> A407SK - 4 </td><td></td></tr><tr><td>盐酸</td><td>费舍尔</td><td> A142 - 212 </td><td></td></tr><tr><td> Percoll </td><td> GE医疗集团</td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td>费舍尔</td><td> P285 - 500 </td><td></td></tr><tr><td> PierceSDS - PAGE样品制备试剂盒</td><td>刺穿</td><td> 89888 </td><td></td></tr><tr><td>氯化钠</td><td>零售物价指数</td><td> S23020 </td><td></td></tr><tr><td>碳酸氢钠3 </td><td>费舍尔</td><td> BP328 - 500 </td><td></td></tr><tr><td>娜2保护海港条例"4 </td><td>费舍尔</td><td> S381 - 500 </td><td></td></tr><tr><td>蔗糖</td><td>零售物价指数</td><td> S24060 </td><td></td></tr><tr><td> Tris碱</td><td>零售物价指数</td><td> T60040 </td><td></td></tr><tr><td>河豚毒素</td><td>西格玛</td><td> T5651 </td><td></td></tr><tr><td>快临标签混合S35简易标签</td><td> Perkin Elmer公司</td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th>设备</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>解剖工具</td><td></td><td></td><td></td></tr><tr><td> Dounce匀浆，7毫升（带有两个玻璃杵标记为"A"和"B"） </td><td>惠顿</td><td></td><td></td></tr><tr><td> P1000的吉尔森Pipetman </td><td>吉尔森</td><td> F123602 </td><td></td></tr><tr><td> Allegra的6KR离心机</td><td> Beckman Coulter公司</td><td> 366830 </td><td></td></tr><tr><td>生长激素3.8转子，水平转子</td><td> Beckman Coulter公司</td><td> 360581 </td><td></td></tr><tr><td>贝克曼J2 - 21型离心机</td><td>贝克曼</td><td></td><td></td></tr><tr><td>贝克曼管与帽</td><td>贝克曼</td><td> 355672 </td><td></td></tr><tr><td>白色围墙适配器</td><td>贝克曼</td><td> 342327 </td><td></td></tr><tr><td>蓝壁适配器</td><td>贝克曼</td><td></td><td></td></tr><tr><td> JA - 17转子，固定角转子</td><td>贝克曼</td><td> 369691 </td><td></td></tr></tbody></table>用于免疫印迹抗体表: <table border="1"><tbody><tr><th>抗体名称</th><th>公司</th><th>目录编号</th><th>宿主物种</th><th>稀释倍数</th></tr><tr><td> β-肌动蛋白</td><td>西格玛</td><td> A5441 </td><td>小鼠</td><td> 1:2000 </td></tr><tr><td> GFAP </td><td>圣克鲁斯</td><td> SC - 65343 </td><td>小鼠</td><td> 1:200 </td></tr><tr><td> GP73 </td><td>圣克鲁斯</td><td> SC - 134509 </td><td>兔</td><td> 1:200 </td></tr><tr><td> HSC70 </td><td>圣克鲁斯</td><td> SC - 7298 </td><td>小鼠</td><td> 1:200 </td></tr><tr><td> Laminβ </td><td>圣克鲁斯</td><td> SC - 6261 </td><td>山羊</td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td>圣克鲁斯</td><td> SC - 28259 </td><td>兔</td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore公司</td><td> MAB1596 </td><td>小鼠</td><td> 5微克/μL </td></tr><tr><td> SNAP25 </td><td> Abcam公司</td><td> ab5666 - 100 </td><td>兔</td><td> 1:2000 </td></tr><tr><td>突触</td><td> Millipore公司</td><td> MAB368 </td><td>小鼠</td><td> 1:500 </td></tr><tr><td> β- 3 -微管蛋白</td><td>圣克鲁斯</td><td> SC - 80016 </td><td>小鼠</td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

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。

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

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

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。

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 ...

preparation-synaptoneurosomes-from-mouse-cortex-using-discontinuous

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

31.4K 次观看.  University of Wisconsin. 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。

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

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

生物学

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

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

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 ...

神经科学

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

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 ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

使用多体分析在发育中的鼠标大脑中进行翻译分析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

亚细胞分离用于从小鼠脑中分离突触成分

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

Mononuclear Cell Isolation from Mouse Brain: A Density Gradient Centrifugation Technique to Isolate Brain Resident Mononuclear Cells

Source: Ji, Z. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/61050/flow-cytometric-analysis-lymphocyte-infiltration-central-nervous">Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis.</a> J. Vis. Exp. (2020)
In this video, we perform the isolation of mononuclear cells from a mouse&#8217;s brain using density gradient centrifugation. The isolated mononuclear cell suspension can be used for further downstream analysis.

从小鼠脑中分离单核细胞:一种分离脑驻留单核细胞的密度梯度离心技术

Source: Ji, Z. et al. Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis. J. ...

JoVE Encyclopedia of Experiments

癌症研究

Cancers of the Nervous System

Ex vivo studies

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Source: Gottschling, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54757">The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions.</a>&#160;J. Vis. Exp.&#160;(2016)
This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

一种用于研究神经元-胶质细胞相互作用的间接神经元-星形胶质细胞共培养技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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