这项工作是由加拿大卫生研究院的研究MOP-81158的补助金。

1。从原代培养海马神经元的目标蛋白的提取<ol><li>在收获前从海马细胞内源性蛋白，准备与蛋白酶抑制剂的裂解缓冲液（LB， 表1）。至10毫升的裂解缓冲液中，加入0.1毫升苯甲基磺酰氟溶液甲基磺酰氟（PMSF）至终浓度为10mM，并添加1蛋白酶抑制剂鸡尾酒片剂。 </li><li>制备N-乙基马来酰亚胺（NEM）的溶液（ 表1）从冻干粉末。轻轻溶解在100％乙醇溶液中，在室温（RT）。一定要准备NEM的解决方案，新的一天的实验。 </li><li>接下来结合的裂解缓冲液和NEM。 2M的的NEM解决方案，到一个新的50毫升锥形管中加入0.5毫升。在顶部加入20毫升LB，并迅速到位LB +终浓度为50mm（NEM）在4°C摇摆的平台，充分搅拌5分钟。总是添加NEM / EtOH溶液的第一和LB的第二个，这样才能适当地混合使用这两种。 </li><li>从孵化器和冰*上删除培养的神经元。轻轻地取出细胞介质，并轻轻地洗两次，用冷磷酸盐缓冲液（PBS）1个。加入300毫升的LB + 50mM的NEM的海马神经元，刮细胞，用一次性电池升降机的第一口井。收集的细胞裂解液，然后放电到该组的下一个孔中的溶胞产物。刮取细胞内的第二口井使用的细胞升降机，收集和重复使用，三分之一好了，如果需要进一步集中的裂解。 </li><li>到预冷却的（冰浴），1.5 ml离心管中收集细胞裂解液。重复步骤1.4用100μl的LB + 50mM的NEM收取任何剩余的细胞裂解液。通过总细胞裂解液通过26½计注射器5-6次，以帮助机械溶解，小心不要吹泡泡的解决方案。相反，人们可以在冰上超声，持续15秒，如果设备是可用的。章动≥30分钟的细胞裂解物在4℃下</li><li>澄清的细胞lysA的“特以16,000 xg离心/ 30分钟，通过离心分离，以沉淀未溶解细胞和洗涤剂的不溶性材料。 </li><li>收藏报错清零的细胞裂解液（上清液）在一个新的预冷的1.5毫升管在冰上。重复各组细胞的收获协议。在所有裂解液样品，用BCA检测，测量蛋白质浓度和规范组细胞裂解样品的量正好等于总蛋白，建议最低为500微克。把所有样品的总体积500μLLB + 50mM的NEM。 </li><li>将1-5微克针对靶蛋白的第一抗体添加到每个溶胞产物样品。章动的抗体溶胞产物混合样品过夜，在4℃下</li></ol> *小学大鼠海马神经元应当在约250,000个细胞的密度培养/孔在6孔培养皿中，在无血清培养基中含有B27的神经元的补充，如先前描述的5星 ，和生长的欲望d长度的时间，通常为14-16天， 在体外 （DIV），以达到成熟。建议至少500μg总蛋白成功地免疫沉淀和biotinylate的目标神经元蛋白，这通常需要2-3井的6孔培养皿。 2。沉淀的抗体结合的靶蛋白<ol><li>在目标蛋白质的沉淀，固定，准备浆蛋白，或蛋白涂G-Sepharose磁珠50％。具体而言，≥60μl的每个样品的琼脂糖珠添加1.5 ml离心管，确保所有的样品有等量的珠子。磁珠也适用，如果设备是可用的。 </li><li>加入等体积的50％的淤浆到每个抗体溶解物的样品，和章动≥1小时，在4℃下</li></ol> 3。酰基-生物素交易所：羟胺（HAM）裂解<ol><li>在执行步骤2.2，用裂解缓冲液（LB）的二制备的管数fferent pH值。 pH值是很重要的，这些步骤应始终使用pH计进行调整。准备2毫升的LB pH为7.2％的样品，严格的缓冲器，每个样品（ 表1）和0.5毫升。还要准备0.5毫升LB + 10mM的NEM每个样品的步骤1.1-1.3。加入PMSF和蛋白酶抑制剂片所有的裂解缓冲液，在步骤1.1。 </li><li><ol class="lalpha"><li>羟胺（HAM）是一个功能强大的还原剂，其裂解棕榈酸所需的生物素化（ 图1）从半胱氨酸，和遗漏的HAM卵裂用作阴性对照。斯普利特到一个省略HAM裂解步骤（HAM），和一个包括HAM工序（+ HAM）的两个样本，每个样本的珠。为了标准化HAM治疗引起的蛋白质降解，1应该每个样品拆分成的三分之二，用1/3-HAM控制用于珠粒，和用于+ HAM处理中剩余的三分之二。 </li><li>准备额外的1。5 ml离心管上的冰，作为-HAM控制，每个样品标记。步骤2.2，0.5 XG / 1分钟，轻轻离心所有样品的珠子，在4°C（离心机以这样的速度，持续时间和温度除另有注明外，所有剩余的步骤），将管上的冰，除去上清液，在600μl的LB + 10毫米NEM重悬珠。 </li></ol></li><li>重新悬浮后的样品在裂解缓冲液+ NEM 10mM的珠，立即收集200μl的样品的溶解物珠淤浆，和排出到该样品-HAM管上冰。添加一个额外的300微升的LB + 10mM的NEM-HAM样品，和一个额外的100μl到+ HAM样本的每个样品中加入500μl共有。孵育管在冰上10分钟。 </li><li>重新挂起（洗）-HAM + HAM样本轻轻地用严格缓冲0.5毫升，每个样品。快速执行此步骤，作为一个较长的SDS孵育将导致从结合的抗体中除去珠。 </li><li>所有样品轻轻地洗三次在0.5毫升/ LB样品的pH值7.2，和自旋向下的清洗在步骤32亿之间。 </li><li>在执行步骤3.5的同时，准备的HAM缓冲液（ 表1）。适当体积的羟胺溶液加入到新的管中，以达到最终浓度为1M的在0.5毫升LB培养基，pH为7.2，每+ HAM样品的体积。一定要准备新的一天的实验HAM缓冲。加入0.5毫升/样品的LB pH值7.2-HAM的样品，和0.5毫升/样品，HAM的缓冲区全部+ HAM样本。 </li><li>章动所有样品1小时，在室温（RT）。不要超过1小时。 </li></ol> 4。酰基-生物素交易：生物素-BMCC标签<ol><li>在执行步骤3.6中，准备2毫升的LB pH值6.2％的样品（ 表1），在步骤3.1。将LB pH值为6.2，在冰上，直到需要。还要准备一个原液生物素-BMCC（ 表1）在使用前。称取exacTLY 2.1毫克生物素-BMCC，收集在1.5 ml管中，并温和地溶解在0.5 ml二甲基亚砜（DMSO），在室温下放置在摇摆平台，实现的8MM浓度（不使用吸管，打破了粉）。 </li><li>步骤3.6完成后，轻轻地洗磁珠一次在pH值6.2的裂解缓冲液，以去除残留的HAM的缓冲。除去上清液，并在冰上放置样品。 </li><li>生物素-BMCC是maliemide的的免费的半胱氨酸的硫醇基团（ 图1）具有高亲和性的结合生物素分子。当执行步骤4.2，每个样品的制备生物素-BMCC缓冲液的0.5毫升的（ 表1），在工作浓度0.5μM，5μM之间。超过5μM的浓度可能会饱和靶蛋白6，并且通常不应该超过，然而，这可能会有所不同，这取决于所需的目标蛋白。每个样品中加入0.5毫升的生物素-BMCC缓冲，和章动了整整1小时，4＆度; C.不要超过1小时。 </li></ol> 5。生物素共轭的目标蛋白和SDS-PAGE洗脱<ol><li>步骤4.3一旦完成后，轻轻地清洗所有样品的在LB pH值6.2。保留所有样本冰之间的清洗。删除没有还原剂（ 表1）从-20℃存储2×SDS样品缓冲液，并慢慢地在冰上解冻。 </li><li>轻轻冲洗样品三次LB pH值7.5 + PMSF /抑制剂，冰之间的清洗，并保持样品。 </li><li>第三次洗涤除去上清液，留下沉淀的珠在浆液中，该管的底部与剩余缓冲器。最底层的料浆管，通过一个吸管蘸小直径端（凝胶加载尖或P2移液器吸头就足够了），并迅速收集所有剩余的缓冲，要小心，不要采取任何珠。 </li><li>补编与DL-二硫苏糖醇（DTT）的2倍的SDS样品缓冲液，以达到最终浓度为5mM的。加40-50μl的2×上样缓冲液+ 5mM的DTT到每个样品。如果有必要，涡珠与上样缓冲液混合，并立即在高速离心收集所有的颗粒珠，淹没在上样缓冲液。 </li><li> 10分钟煮沸的所有样品在75-80°C。让样品冷却至室温≥10分钟，台式离心机16000 XG / 3分钟，彻底颗粒的珠子。 </li><li>从每个样品的聚丙烯酰胺凝胶，适用于免疫印迹，使用SDS-PAGE 7内一条单线添增洗脱蛋白的完整的卷（上清液）。组织，使所有样品的HAM控制的单个样品的洗脱剂和+ HAM洗脱液立即运行期间的SDS-PAGE（图2）彼此相邻。 SDS-PAGE后，转移至PVDF或硝酸纤维素膜。 </li></ol> 6。西方杂交和检测目的蛋白的棕榈酰化<ol><li>步骤5.6完成后，洗膜两次在Tris-缓冲盐水（TBS：1倍）上的摆动平台在RT和10分钟。 </li><li>准备一个阻断缓冲液称量出牛血清白蛋白（BSA），以达到3％（重量）的1倍的TBS的体积，以阻断非特异性标记+ Tween-20的0.05％（TBS-T），是足以充分浸没膜。对于链霉亲和素印迹，始终阻止与BSA和奶粉的。摇摆的平台上，阻止膜在室温下1小时。 </li><li>准备TBS-T + 0.3％BSA的抗体，该抗体溶液中。加入链霉亲和素-HRP抗体在1毫克/毫升蒸馏水中，稀释1:5,000-1:10,000重组。一旦完成步骤6.2，一次在TBS-T洗膜10分钟，然后在链霉亲和素的抗体溶液孵育膜上的摇摆的平台要么在室温1小时，或在4℃下过夜</li><li>三次在TBS-T洗膜的摆动平台上10分钟。为了检测的棕榈信号，使用化学暴露HRP发光iluminescent底物试剂盒。 </li><li>为了棕榈酰化水平正常化感兴趣的蛋白进行免疫沉淀的量，使用免疫印迹剥离缓冲区为5-10分钟，在RT上的摆动平台剥离膜。重复步骤6.1 - 6.4使用的主要感兴趣的蛋白质，最初是用于免疫沉淀的抗体的。 </li><li>量化棕榈感兴趣的蛋白用图像分析软件，和棕榈酰化水平正常化免疫沉淀的蛋白的量。 </li></ol>

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没有利益冲突的声明。

IP-ABE分析这里介绍的是一个简单的和高度敏感的检测棕榈酰化蛋白在原代培养海马神经元的方法，主要使用标准生物化学技术。这使得检测实验室已经配备了免疫印迹材料很容易适应。的IP-ABE测定结合了标准的免疫沉淀的协议来隔离和固定的靶蛋白，随后由先前描述的ABE化学2-4棕榈酸对底物蛋白的快速检测水平。 ABE技术具有广泛的应用范围，用于检查棕榈酰化蛋白在各种组织和细胞系，包括大型的棕榈酰-蛋白质组学筛选全球棕榈酰化公司，和棕榈酰化水平的一个单一的靶蛋白的快速检测。 此前的描述ABE化学利用不同的方法，细胞裂解液和洗涤剂提取的神经PROTEINS 2，9，游离硫醇封锁10，11，生物素化，并纯化目标蛋白4，根据应用程序的实验。添加棕榈酸神经元蛋白向细胞膜，在其定位和检测靶蛋白的的棕榈酰化馏分的结果，将需要从这些膜其提取。此前描述的ABE方法已采用了多种离子9和非离子型去污剂8，以提取所需的目标蛋白，和一个特定的洗涤剂的使用依赖于目标蛋白和它的公知的膜贩运。在这里，我们利用非变性和非离子型洗涤剂的IGEPAL CA-630提取棕榈酰化蛋白质，我们已经证实的棕榈酰化神经元蛋白δ-连环蛋白（ 图2）进行提取和检测。 IGEPAL CA-630的结构包括一个笨重和刚性的非极性头部基团不扰乱天然蛋白质的构象或相互作用，但它允许其与膜相关蛋白的疏水区域，从而赋予混溶性并允许其提取。应提取的许多神经元蛋白定位于突触的突触膜或突触后密度，使用IGEPAL CA-630，但是一些可能不完全溶解，并可能需要使用不同的非变性洗涤剂的Triton X-100等，它具有以前用于ABE化学2，8。 ABE化学几个描述信息也利用不同的巯基反应的生物素化试剂分子从我们在这里描述的，称为生物素-HPDP（Thermo Scientific的），这也需要一种不同的蛋白质的纯化手段4。生物素-HPDP是另一个硫醇反应，maliemide的共轭的生物素分子，包含一个二硫键的maliemide连接臂，结构ðifferentiating它从生物素-BMCC，其中不包含此二硫键。巯基-生物素化蛋白质的免费或棕榈酰化半胱氨酸生物素-HPDP之后，在得到的二硫化键的目标蛋白和的生物素基团之间可以通过减少试剂到释放的生物素基团和再生的未改性的形式的蛋白质裂解，使生物素化通过生物素-HPDP短暂的，可逆的。因此，通过使用该生物素化试剂需要纯化靶蛋白的固定化的抗生物素蛋白的试剂，如链霉亲和素包被的磁珠。然而，硫醇 - 生物素化靶蛋白的生物素-BMCC，在这里，我们描述的，是稳定的和相对较永久，需要净化的靶蛋白的抗体，该抗体针对的目标，并固定在琼脂糖珠。两个ABE技术此前已用于大型屏幕，和棕榈单一蛋白质2，8-10，12的检测，和IP-ABE的分析，我们在这里描述的是最快速检测单个神经元的靶蛋白的棕榈。 绝大多数的细胞棕榈涉及可逆的棕榈半胱氨酸的巯基（称为S-棕榈，我们概括为“棕榈”，在此协议），但是非常有限的一组蛋白质是不可逆的棕榈甘氨酸和半胱氨酸残基通过形成稳定的酰胺键，称为N-棕榈酰化13。 ABE化学的一个限制是它无法检测N-棕榈酰化，由于酰胺键的稳定性，应确认等棕榈酰化的一种新的目标蛋白的筛选使用3H-棕榈酸代谢标记1。 正如前面所提到的，可以很容易地适应的IP-ABE检测检查棕榈酰化在来自多个源的蛋白提取物中，包括转录sfected异源细胞系中，基层组织，主要从多个脑区的神经细胞。 IP-ABE法已被用于研究突变的半胱氨酸-丝氨酸蛋白的棕榈酰化半胱氨酸找准位置沿靶蛋白8的棕榈充足，基础条件下培养的神经元突触蛋白的研究动态棕榈营业额10，和变化后的神经元蛋白的棕榈酰化水平在诱导的神经元的电活动9。的IP-ABE检测的敏感性和适应性使其非常适合于研究棕榈配置文件的神经元蛋白，最适用于检测动态变化棕榈。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> IGEPAL CA-630 </td><td>西格玛</td><td> I8896 </td><td></td></tr><tr><td> PMSF </td><td> Fluka公司</td><td> 93482 </td><td></td></tr><tr><td>蛋白酶抑制剂</td><td>罗氏公司完整的迷你</td><td> 11 836 153 001 </td><td></td></tr><tr><td> NEM </td><td>西格玛</td><td> E3876 </td><td></td></tr><tr><td> HAM解决方案</td><td>西格玛</td><td> 46780-4 </td><td></td></tr><tr><td> BCA蛋白定量试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td><td>确保与选择洗涤剂的兼容性</td></tr><tr><td>蛋白G /涂A-Sepharose磁珠</td><td> GE医疗集团</td><td> 17-6002-35 </td><td></td></tr><tr><td>生物素-BMCC </td><td> Thermo Scientific的</td><td> 21900 </td><td></td></tr><tr><td>链霉亲和素-HRP </td><td> Thermo Scientific的</td><td> 21126 </td><td>复溶在1毫克/毫升在水中</td></tr><tr><td> Western Blot法溶出缓冲区</td><td> Thermo Scientific的</td><td> 21059 </td><td></td></tr></tbody></table>

detection of protein palmitoylation in cultured hippocampal neurons by immunoprecipitation and acyl-biotin exchange (abe)

棕榈酰化是转译后的脂质变形例涉及的附件通过不稳定的硫酯键的底物蛋白质的半胱氨酸残基的16 -碳饱和脂肪酸，棕榈酸，[1]评论中。棕榈酰化的基板蛋白增加其疏水性，通常有利于其贩运朝向细胞膜。最近的研究表明棕榈是最常见的脂质修改，神经元1，2，，棕榈营业额是一个重要的机制，这些细胞的蛋白质调控的针对性和贩运。因此，更好地识别和检测的棕榈酰化基体的我们的理解贩卖的蛋白质在神经元。 蛋白质棕榈酰化在过去已检测技术上的阻碍，由于缺乏底物蛋白序列之间的共识，并依赖代谢labeling的棕榈酰蛋白与3 H-棕榈酸酯，一个耗时的生化检测灵敏度低。酰基-生物素交易所（ABE）检测使开发更快速和高灵敏度检测的棕榈酰化蛋白质2-4，是最优的，用于测量对神经元的蛋白质，棕榈酸的动态营业额。的ABE测定是由三种生化状态的步骤（ 图1）：1）不可逆封锁未修改的半胱氨酸的硫醇基团的使用N-ethylmaliemide（NEM），2）的特异性裂解和去掩蔽的棕榈酰化的半胱氨酸的硫醇基羟胺（HAM），和3）选择性标记的棕榈酰化的半胱氨酸，使用巯基反应的生物素化试剂，生物素-BMCC。 ABE步骤巯基的生物素化蛋白的纯化不同，这取决于实验的总体目标。 在这里，我们描述了一种方法，棕榈酰化蛋白的纯化主要海马由初始免疫沉淀法（IP）的步骤中使用的蛋白质，随后由ABE法和免疫印迹，以直接测量棕榈酰化，蛋白质的水平，这被称为IP-ABE检测抗体，该抗体针对的人神经元。低密度培养的胚胎大鼠海马神经元已被广泛用于研究的神经元蛋白的定位，功能和贩运，因此非常适合使用IP-ABE分析，为研究神经元蛋白棕榈酰化。的IP-ABE检测主要是要求标准的IP和蛋白质印迹试剂，并且仅受限于目标衬底的可用性抗。此法可以很容易地适用于在异源细胞培养，主要来自不同的小鼠和大鼠脑组织神经细胞，甚至是主要的脑组织本身的转棕榈酰化蛋白的纯化和检测。

的IP-ABE检测特异性检测沿底物蛋白质的半胱氨酸残基的硫醇-棕榈酰化，并且可以被用于检测在图1中所示的方式中的免疫沉淀的神经元蛋白棕榈酰化。治疗神经元的免疫沉淀的蛋白质（ 图1）的HAM切割棕榈酸和半胱氨酸的硫醇基之间的硫酯键，使生物素-BMCC具体掺入到新的可用的硫醇基团，随后可以使用蛋白质印迹法检测。遗漏HAM（负HAM）裂解的防止掺入生物素-BMCC和作为阴性对照的棕榈酰-生物素化的加-HAM样本为特定的功能，因此，应总是运行相邻的加-HAM样本对西方印迹通过SDS-PAGE（ 图2）。 在一个优化的实验（ 图2A），棕榈酰化的神经元蛋白δ-连环蛋白2的信号可以容易地通过杂交的生物素-BMCC在浓度为1μM时，使用链霉抗生物素蛋白与辣根过氧化物酶共轭的，对平行的负加HAM样品检测。棕榈酰化的特定δ-连环蛋白的信号出现在它的预测大小160KD，只加-HAM样本。这个信号的特异性确认通过与特定的抗体，该抗体最初被用于免疫沉淀，所得的信号中预测的相同的大小在两个的HAM治疗在reprobingδ-连环蛋白。优化的IP-ABE吸附试验（ 图2A），将导致一个减号-HAM的样品，这是易于区分的加-HAM样品中的蛋白质印迹的档案，并表现出很小，没有棕榈酰化信号。 用于标记棕榈酰化的半胱氨酸的浓度的生物素-BMCC需要仔细优化，并且如果超饱和浓度的双向otin-BMCC期间使用的ABE化学步骤（ 图2B），过高的背景和非特异性信号将是可见的。先前已被确定为生物素化的神经元蛋白一个棕榈酰化，α7烟碱样乙酰胆碱受体，使用生物素-BMCC甲线性响应曲线6，并显示在5-10μM的浓度接近完全饱和。虽然会偏离最佳浓度的生物素-BMCC上略有不同的神经元靶蛋白，治疗用生物素-BMCC在超过5μM的浓度可能会超饱和的目标蛋白，并在免疫印迹的档案结果可见背景和非特异性信号。浓度为0.5μM的生物素-BMCC以前用于检测其他神经元蛋白的棕榈，包括SNAP-25，亨廷顿，AMPA受体亚基GluA1和GluA2，和paralemmin 8，并测定了一种新的最佳浓度为靶蛋白可以通过试验和错误，以线性的方式来完成的，从0.5-5μM，我们在这里描述的范围内开始。所得的链霉抗生物素蛋白免疫印迹的公司之间的的负加HAM样本δ-连环蛋白棕榈酰化4μM的生物素-BMCC（ 图2B），与附加的背景信号，以不同的大小，这表明发生了不合适的生物素化的处理时，会出现类似的。 羟胺是一种强大的还原剂，在除了其高效的硫酯键的裂解的靶蛋白的降解的有限的结果。因此，ABE化学的优化必须正常化负加HAM样本（步骤3.2 - 3.3）用于免疫沉淀的蛋白质的量。正常化需要使用双重加对比HAM负的样品，这实际上导致无法区分韦斯特固定化靶蛋白的量Ň印迹公司的靶蛋白，δ-连环蛋白（ 图2A，B）所示。然而，如果固定化靶蛋白的量未标准化负加HAM样本之间，可以得到的免疫印迹的信号，用于靶蛋白中的加-HAM样本丢失，所示为δ-连环蛋白时，等量的蛋白被用来在两个HAM的处理（ 图2C）。 棕榈酰化蛋白标记的特异性ABE化学是非常敏感的，需要优化。过程中遇到的IP-ABE检测常见的陷阱，包括了次优的结果示于图2B和2C，和退化的目标蛋白，HAM的治疗，这通常是由较大的试剂和缓冲液，和不正确的pH值无关。为了避免这些缺陷，并纠正子最优ABE化学的，新鲜度和所用试剂的浓度所需的表1中列出的缓冲区应始终进行检查，并应始终检查运行实验之前的所有缓冲器的pH值调整。 <table border="1"><tbody><tr><td colspan="2"> 缓冲区 </td><td> 使用浓度 </td><td> 评论 </td></tr><tr><td> 裂解缓冲液（LB） </td><td>在蒸馏H 2 O：1％的IGEPAL CA-630的50mM的Tris-HCl pH7.5的150mM NaCl的10％甘油</td><td> N / A </td><td>实验前准备和存储在4°C </td></tr><tr><td> NEM解决方案 </td><td>在100％乙醇：NEM冻干粉</td><td> 2 M </td><td>准备新鲜的，在使用之前。 </td></tr><tr><td> 严格的缓冲区 </td><td>在LB：10mM的MEM 0.1％SDS中</td><td> N / A </td><td>准备不久在使用前，保持在冰上。 </td></tr><tr><td> LB pH值7.2 </td><td> LB：调整PH准确7.2 </td><td> N / A </td><td>使用pH计在即将使用前将pH调节。 </td></tr><tr><td> HAM缓冲区 </td><td>在LB pH值7.2：联合HAM解决方案</td><td> M（最后HAM浓度） </td><td>使用前准备。 </td></tr><tr><td> LB pH值6.2 </td><td> LB：调整pH值，以准确6.2 </td><td> N / A </td><td>为LB pH值7.2相同</td></tr><tr><td> 联合生物素-BMCC解决方案 </td><td>在DMSO：2.1毫克生物素-BMCC解决方案</td><td> 8毫米</td><td>使用前准备。 </td></tr><tr><td> 生物素-BMCC缓冲区 </td><td>在6.2 LB pH值：添加生物素-BMCC的解决方案</td><td> 1 - 5μM（最终浓度生物素-BMCC） </td><td>使用前准备。 </td></tr><tr><td> 2×SDS样品缓冲液，无还原剂</td><td>在蒸馏H 2 O：5％SDS 5％甘油125MM的Tris-HCl pH 6.8的0.01％溴酚蓝</td><td> N / A </td><td>使用前实验，并存储在-20°C至准备。补充DTT在使用前用5毫米。 </td></tr></tbody></table> 表1中。所需的缓冲液和试剂为IP-ABE测定。许多的裂解缓冲液，可以制备开始之前的实验，但是进行检查和调整pH值应在使用前立即用pH计，以确保成功的ABE化学。大多数股票的解决方案应新鲜配制，为每一个实验，在使用之前。许多缓冲器需要试剂共同大多数实验室配备用于生物化学，并与供应商信息的特种试剂的试剂和设备的表中列出。 图1。示意性的免疫沉淀和在酰基和素-生物素Exchange（IP-ABE）检测，纯化和检测的神经元蛋白的棕榈酰化。裂解培养的海马神经元，（1）的目标蛋白，然后纯化，使用靶特异性抗体，并固定在琼脂糖凝胶珠涂覆有蛋白质G或A，将纯化目标蛋白质是然后（2）用N-ethylmaliemide（NEM）的处理，不可逆地结合并阻止沿未修改的半胱氨酸（C）的游离巯基（-SH）基。靶蛋白，然后（3）进行治疗与羟胺（HAM），导致在特定的硫酯键裂解棕榈酰化的半胱氨酸和免费的棕榈酰化硫醇基（-SH）的揭露。接着，对靶蛋白是（4）汤治疗d与巯基的反应性的生物素分子，生物素-BMCC，从而在特定的生物素化的棕榈酰化的半胱氨酸。最后，（5）的生物素化的靶蛋白被洗脱并除去从抗体及胎圈。的目标蛋白与生物素标记的棕榈酰化半胱氨酸（S）是适用于SDS聚丙烯酰胺凝胶电泳（SDS-PAGE），并与链霉亲和素的免疫印迹检测纯化的神经元蛋白的棕榈。 <a href="https://www.jove.com/files/ftp_upload/50031/50031fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50031/50031fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50031/50031fig2highres.jpg"/> 图2。检测使用的IP-ABE测定的神经蛋白δ-连环蛋白的棕榈酰化。原代大鼠海马神经元生长作为预viously描述5星 ，在触发信息，或者最多的密度为130个细胞/ mm 2，直到到期，然后裂解，并经受到IP-ABE检测到检测最近确定的的棕榈酰化神经元基板，δ-连环蛋白2的棕榈。纯化的免疫沉淀（IP地址）的δ-连环蛋白（目标蛋白质）使用5μg的每个样品的δ-连环蛋白抗体（BD Transduction Laboratories公司）进行分离，并进行SDS-PAGE，然后进行并行减，加羟胺（HAM）样本。通过蛋白质印迹法（WB）与链霉亲和素-HRP（棕榈酰化）检测棕榈酰化，和膜被剥离并进行到一个重新探测WBδ-连环蛋白。 （A）的一个优化的代表IP-ABE结果棕榈酰化的δ-连环蛋白（箭头）生物素-BMCC用1μM处理，示出ABE化学检测来自IP样本硫醇棕榈酰化蛋白的特异性。 （B）一个次优的representative IP-ABE结果δ-连环蛋白，其中使用了过量浓度的生物素-BMCC4μM的，从而导致在非特定的信号和背景在两个负加HAM样品（箭头）。 （C）的另一种次优的代表IP-ABE WBδ-连环蛋白，其中的过度4μM的浓度的生物素-BMCC使用，以及用于加-HAM IP样品的蛋白质的量没有归一化的HAM-介导的蛋白质降解，从而导致在重新探测WBδ-连环蛋白在丢失信号。 <a href="https://www.jove.com/files/ftp_upload/50031/50031fig2large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE at JoVE.com

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

棕榈酸与蛋白质的可逆加成是一个重要的调节细胞内蛋白质的活动。许多突触蛋白被棕榈酰化的神经元中，这是一个特别令人感兴趣。我们利用一个简单的检测棕榈酰化蛋白在培养的神经元，这可适于多种细胞类型和组织的生化方法。

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

detection-protein-palmitoylation-cultured-hippocampal-neurons

Research

JoVE Journal

Neuroscience

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

35.2K 次观看.  University of British Columbia. 棕榈酸与蛋白质的可逆加成是一个重要的调节细胞内蛋白质的活动。许多突触蛋白被棕榈酰化的神经元中，这是一个特别令人感兴趣。我们利用一个简单的检测棕榈酰化蛋白在培养的神经元，这可适于多种细胞类型和组织的生化方法。

视频: Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.