Name: immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins
Uploaded: 2016-03-03T14:00:00.000+00:00
Duration: 5 min 35 s
Description: Watch this Scientific Journal Video about Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins at JoVE.com

这项工作是由美国国立卫生研究院资助GM68418的支持。

1.细胞培养和转染<ol><li>放的盖玻璃（22毫米×22毫米）在6孔聚苯乙烯板。可选的外套盖玻璃，聚L-赖氨酸，0.1％w / v的，水（见材料/设备清单），以保证有丝分裂细胞上按照以下步骤玻璃盖: 注意:最佳条件，必须对每个细胞系和应用来确定。 <ol><li>用聚-L-赖氨酸，0.1％重量/体积，在无菌条件下涂层培养表面在水（0.4mL毫升/孔的6孔聚苯乙烯板）。轻轻摇动，以确保即使在培养表面涂层。 </li><li> 5分钟后，吸删除解决方案，并彻底冲洗用无菌组织培养级别的水面。 </li><li>引入细胞和中干前至少2小时。 </li></ol></li><li>种子HeLa细胞17或HeLa细胞的Tet-Off细胞上放置在6孔聚苯乙烯板的盖玻璃（22毫米×22毫米）17,22。检查细胞密度为5.4×10 5每孔。培养细胞在具有10％FBS和1％青霉素 - 链霉素的高葡萄糖DMEM中。 注意:为了达到最佳效果，凭经验确定播种用的细胞密度。 </li><li>孵育在37℃下将细胞在5％的CO 2的气氛中18小时。 注意:在HeLa细胞的Tet-Off细胞的情况下，外源基因的转录是在不存在诱导剂（ 即 ，四环素/多西环素），因此培养细胞无四环素/多西环素的活性，并与pTRM4表达载体（ 表2瞬时转），其转录由TRE启动子调控（见下文）。 </li><li>播种，转染细胞18小时后，如下所示: <ol><li>通过混合1.5微升的siRNA寡使溶液A（20μM的退火的库存; 表1）和/或2.0微克质粒（ 表2）在50微升减少血清的培养基（参见材料/设备的列表），并在室温孵育5分钟。 注意:在此分析中，CA-UTR的siRNA（5&#39;3和&#39;非编码区的siRNA的混合物; 表1）共转染在协议3和4（见讨论）一起使用。 </li><li>通过混合0.75微升转染试剂I（参见材料/设备的列表）在50μl降低血清培养基，并在室温孵育5分钟使B液。 注:可选的步骤是增加了1.0微升转染试剂II（见材料/设备的列表）。 </li><li>混合溶液A和B一起，并在室温孵育15分钟。 </li><li>用PBS洗涤培养细胞一次，然后加入500μl血清培养基减少到6孔聚苯乙烯板的各孔中。添加溶液A和B（ 即 ，RNA和/或DNA -脂质复合物）的混合物中直接向每个单独的井的。 注意:终浓度为3.3微克/毫升（质粒）; 50纳米（siRNA）的。 </li><li>孵育在37℃下将细胞在5％的CO 2的气氛中4.5小时。介质更改为高糖DMEM机智H 10％FBS和1％青霉素 - 链霉素。 </li><li>孵育在37℃下将细胞在5％的CO 2的气氛中对转染后48-72小时。 注意:对于最佳的结果，固定之前的潜伏期为细胞生长，必须根据经验来确定，和蛋白质消耗和/或表达，必须通过Western印迹分析来确认（参见协议6）。 </li><li>如果有丝分裂细胞分析很感兴趣，固定前添加紫杉醇（10纳米），以培养细胞24小时，或固定前添加TN16（0.5μM）到培养细胞2.5小时。 </li></ol></li></ol> 2.细胞固定和免疫荧光染色检测内源性着丝粒着丝粒蛋白（多聚甲醛固定） <ol><li>固定剂，缓冲液和试剂的制备。 <ol><li>新鲜制备50毫升的在PBS，pH 7.4的4％低聚甲醛溶液中。 <ol><li>加40毫升1-×PBS中至玻璃烧杯中在搅拌板在通风橱。热而stirring至约60℃。 2克低聚甲醛粉末添加到加热的PBS中。 </li><li>通过在总加入1ml的1N NaOH提高缓慢的pH值，因为粉末不会立即溶解。 注:加入NaOH后的溶液清除。 </li><li>一旦多聚甲醛溶解，冷却，滤出液。 </li><li>调节pH值用1N HCl至pH 7.4，并将该溶液用1×PBS的体积至50ml。 注意:溶液的等分试样可以冷冻或贮存在2-8℃下，只要1周。溶液应冰冷或储存在4℃下直到它被使用。 </li></ol></li><li>制备50ml的缓冲液KB1，它由10毫摩尔Tris盐酸，pH为7.5的; 150毫摩尔NaCl; 0.5％BSA;和0.5％的Triton X-100。 注:BSA应新鲜添加。 注:该缓冲区KB系列是基于以前的报告中描述的缓冲KB 1,2,4。 </li><li>制备50ml的缓冲液KB2，它由10毫摩尔Tris-HCl中的，pH为7.5; 150毫摩尔NaCl;和0.5％的BSA。 注:BSA应新鲜添加。 </li><li>制备1ml含有DAPI（50毫微克/毫升）缓冲KB3的。 </li><li>制备100毫升的安装平台，它由1毫克/毫升对苯二胺的; 10％的PBS，90％甘油。 <ol><li>调整为1×PBS中，以9.8的pH值用1N NaOH，在溶液中溶解对苯二胺，然后添加甘油。 </li><li>商店在-80℃1ml等份。避光。 </li></ol></li></ol></li><li>在48-72小时后转染（见方案1），进行细胞固定除去通过抽吸培养基中。冲洗细胞一次，用PBS。应用PBS到培养孔的侧面，以避免干扰所述细胞的表面上。 注:细胞固定的最佳时间点，必须根据经验来确定。 </li><li>固定在PBS中的4％多聚甲醛将细胞在4℃下30分钟。冲洗细胞两次用缓冲液KB2充分地除去残留的4％多聚甲醛。 </li><li> Permeabi丽泽在缓冲器KB1样品在室温30分钟。用缓冲液KB2冲洗细胞一次，并在室温添加缓冲KB2 5分钟以非特异性结合的附加块的站点。 注:缓冲KB1也显著有助于阻止。 </li><li>从使用镊子6孔聚苯乙烯板卸下的盖玻璃（22毫米×22毫米）。使用疏水屏障笔（见材料/设备清单），画一个浅绿色的方形或圆形周围形成每个盖玻璃样品疏水性障碍。请勿触摸或太靠近细胞与疏水屏障笔。安装了新的6孔聚苯乙烯板玻璃盖。 </li><li>稀释的初级抗体对丝粒或动粒蛋白（第1稀释比:100到1:200;也参见材料/设备的列表）和任一抗CENP-B抗体（第1稀释比:400）或抗着丝点抗体（ACA）（1稀释比例:2,000）作为缓冲KB2着丝粒的位置标记。 注意:为了获得最佳效果，最终concentra在该溶液中的一级抗体的灰必须凭经验确定。 </li><li>应用稀释的初级抗体的足够的体积（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞用缓冲KB2 3次。 </li><li>用缓冲KB2，稀荧光团缀合的第二抗体（第1稀释比:200:100:1）针对每个第一抗体。 注意:为了获得最佳效果，二次抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>施加足够的体积将稀释的次级抗体（ 约 30微升在盖玻璃的下降）到浸入细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞5次缓冲KB2期间的30分钟（次6分钟洗涤）。 注意:为了达到最佳效果（ 即对细胞的损失最小），最佳的洗涤条件必须确定empiricaLLY。 </li><li>应用含有DAPI（50毫微克/毫升），以浸没细胞样品缓冲KB3的足够体积。在室温下孵育5分钟的细胞样本。冲洗细胞用缓冲KB2 1-2次。 </li><li>装入包含细胞样品到微滑动玻璃盖。 <ol><li>放置在微滑动的中心安装介质的压降。 </li><li>从细胞样品中移除液体，并使用手或镊子，在微滑动的中心样本位置。避免气泡产生。 </li><li>用纸巾去除多余的封固剂。 </li></ol></li></ol> 3.细胞C端的固定和免疫荧光染色标志标记CENP-A蛋白（丙酮固定） <ol><li>制备<ol><li>准备冰冷的75％丙酮。 </li><li>新鲜制备50ml含0.5％脱脂乳和0.5％BSA的PBS（pH 7.4）中的。 </li><li>新鲜制备50ml含0.1％脱脂乳和0.1％BSA的PBS（pH 7.4）中的。 </li><li>制备1ml的PBS（pH 7.4）中含有DAPI（50-100纳克/毫升）。 </li><li>在2.1.5中描述制备100ml的安装介质。 </li></ol></li><li>在48-72小时后转染（见方案1），进行细胞固定除去通过抽吸培养基中。冲洗细胞一次，用PBS。应用PBS到培养井的侧面，以避免干扰所述细胞的表面上。 注:细胞固定的最佳时间点，必须根据经验来确定。 </li><li>固定在冰冷的75％丙酮的细胞，并孵育细胞在-20℃下10分钟。干细胞在通风橱中在室温下30-60分钟的护罩玻璃。 注意:为了达到最佳效果，需要为固定和细胞干燥的时间长度，必须根据经验来确定。 </li><li>使用疏水屏障笔（见材料/设备清单），画一个浅绿色的方形或圆形周围形成每个盖玻璃样品疏水性障碍。请勿触摸或太靠近细胞与疏水屏障笔。 </li><li>块nonspecIFIC加入含有的PBS 0.5％脱脂乳和0.5％的BSA在室温下5分钟的结合在细胞上的网站。 </li><li>使用含有0.1％脱脂乳和0.1％BSA的PBS稀释的抗Flag抗体（1:1000稀释比），或者抗CENP-B抗体（第1稀释比:200）或ACA（1:2,000稀释比），为着丝粒的位置标记。 注意:为了获得最佳效果，第一抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>应用稀释的初级抗体的足够的体积（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞5次用封闭缓冲期间的30分钟。 注:细胞可以用PBS漂洗。对于最佳结果（ 即 ，为了避免细胞的损失），最佳的洗涤条件，必须根据经验来确定。 </li><li>稀释荧光团缀合的第二抗体（第1稀释比:200:100:1）针对在含有0.1％脱脂乳和0.1％BSA的PBS中的每个主抗体。 注意:为了获得最佳效果，二次抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>应用稀释的二级抗体的足量（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞2次，用含有0.1％脱脂乳和0.1％BSA的PBS。 注意:单独的PBS也可用于洗涤细胞。 </li><li>适用的PBS足够体积含有DAPI（50-100纳克/毫升），以浸没细胞样本。在室温下孵育5分钟的细胞样本。冲洗细胞1-2次，用PBS。 </li><li>安装在2.11中所述含细胞样品到微滑动玻璃盖。 </li></ol> 4.细胞N端的固定和免疫荧光染色标志标记CENP-A蛋白（甲醇固定） <ol><li>制备<ol><li>准备冰冷的甲醇。</li><li>制备的TBS含有4％山羊血清（pH 7.4）中。 </li><li>制备1毫升含TBS DAPI（50-100毫微克/毫升）（pH值7.4）。 </li><li>在2.1.5中描述制备100ml的安装介质。 </li></ol></li><li>在48-72小时后转染（见方案1），进行细胞固定除去通过抽吸培养基中。冲洗细胞一次用TBS。适用的TBS到培养孔的一侧，以避免扰乱细胞的表面上。 注:细胞固定的最佳时间点，必须根据经验来确定。 </li><li>固定在冰冷的甲醇中的细胞，并孵育细胞在-20℃下6分钟。冲洗细胞两次，用TBS充分除去残留的甲醇。 </li><li>使用疏水屏障笔（见材料/设备清单），画一个浅绿色的方形或圆形创建一个围绕每个玻璃罩样品疏水性障碍。请勿触摸或太靠近细胞与疏水屏障笔。 </li><li>块上的CEL非特异性结合位点用含4％山羊血清加入TBS LS。在室温下孵育10分钟。 </li><li>稀释的抗Flag抗体（1:1000稀释）和任一抗CENP-B抗体（第1稀释比:200）:在含有4％山羊血清的TBS（2,000稀释比1）作为着丝粒的位置标记或ACA 。 注意:为了获得最佳效果，第一抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>应用稀释的初级抗体的足够的体积（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞5次用封闭缓冲期间的30分钟。对于最佳结果（ 即 ，细胞的损失最小），最佳的洗涤条件，必须根据经验来确定。 </li><li>稀释荧光团缀合的第二抗体（第1稀释比:200:100:1）在含有4％山羊血清的TBS针对每个初级抗体。 注意:为了达到最佳效果，国际泳联在该溶液中的二级抗体l的浓度必须根据经验来确定。 </li><li>应用稀释的二级抗体的足量（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞3次，用封闭缓冲液。 </li><li>应用含有DAPI（50-100纳克/毫升）浸入细胞样品的TBS的足够体积。在室温下孵育5分钟的细胞样本。冲洗细胞1-2次用TBS。 </li><li>装入包含在2.11中所述的细胞样品到所述微滑动玻璃盖。 </li></ol> 5.免疫荧光图像观察，采集，定量和分析<ol><li>通过配有一个63X和100X油浸镜头，外部紧凑的光源，和一个数字CCD照相机的机动荧光显微镜观察细胞的样品。 </li><li>进行图像采集和处理，包括反褶积，使用软件A，或软件B1和B2（见材料/设备的列表）。请参阅补充编码文件（5.2.1）中的软件A.对于软件B1和B2使用的所有命令使用的所有命令，请参阅（5.2.2）的补充代码文件。 </li><li>用前述方法23-25 ​​量化着丝粒动粒蛋白的信号（ 例如 ，基本维持在着丝粒CENP-A的信号）具有以下稍作修改: <ol><li>在着丝粒动粒蛋白的选择区域内，以及背景如下: <ol><li>有丝分裂细胞:（着丝粒着丝粒区）选择区域与用DAPI染色体重叠;染色体外，但在相同的单细胞内（ 即细胞质区）（背景区域）和面积。 </li><li>相间细胞:（着丝粒着丝粒区）选择区域与染色用DAPI重叠; （背景区）和区外的染色质，但相同的单细胞内（&lt;EM&gt;即细胞内区域）。 注:另见补充编码文件（5.2.1.4.3）或（5.2.2.6.4）的区域选择与软件A或软件B2分别。 </li></ol></li><li>使用软件A或B.对于此任务，使用下面的公式量化的着丝粒剩余信号的百分比: 在着丝粒动粒剩余着丝粒动粒蛋白的信号 <img alt="公式1" src="/files/ftp_upload/53732/53732equation1.jpg" /> 其中s是所选择的区域，这是由ACA或CENP-B染色证实的信号亮度; b是背景信号的亮度; R 样品是用于siRNA（S）处理的细胞的参照ACA或CENP-B信号;和右 Ctrl是参考ACA或CENP-B信号为吕克的siRNA转染的细胞。 注意:在此分析，以前的报告中描述CENP-B信号被用作参考CUL4A信号和RBX1。 <sup&gt; 17 <ol><li>使用这种计算Excel文件复制后从上述信号定量软件粘贴的原始数据。另见补充编码文件:（5.2.1.4）和（5.2.2.6）了解详细信息。计算的一个例子示于表3中 。 </li></ol></li><li>分析至少20个细胞，以消除在染色和图像采集每个测量电平变化。可以选择使用剩余的着丝粒着丝粒信号的"平均值"为每一个分析单元，以这些价值观不同的着丝粒着丝粒蛋白中进行比较。 </li></ol></li></ol>总蛋白的6印迹分析<ol><li>悬浮细胞在变性缓冲液A（20mM的Tris-盐酸，pH为7.4; 50毫摩尔NaCl; 0.5％的Nonidet P-40的0.5％脱氧胆酸盐; 0.5％SDS; 1mM EDTA中;以及完整无EDTA蛋白酶抑制剂混合物）， 26受该悬浮液以声波处理和冻融过程，并测量蛋白质浓度如下: <ol><li>对于超声处理过程中，加50微升缓冲液A从在48-72小时后转染6孔聚苯乙烯板的两井采集的细胞（参见方案1）。操作装有干扰物喇叭和微尖端超声波仪（参见材料/设备的列表），每一个样品合计15秒的间歇脉冲持续时间（占空比50％）。 </li><li>对于冻融过程中，冷冻用液氮细胞，并在室温解冻细胞。 </li><li>使用商业蛋白质检测试剂I或II测量蛋白浓度（见材料/设备的列表）。 注意:裂解物在与任一试剂I或II的蛋白质浓度的测定1:10的比例稀释。在该稀释中，存在于缓冲液A中的SDS示出了在该测定很少或没有干扰。 </li></ol></li><li>混合含有20-30微克总蛋白的裂解物与2×或4×SDS-PAGE上样缓冲液。27煮沸的样品5分钟，然后加载它们在12.0％-15.0％变性电泳SDS-聚丙烯酰胺凝胶。 </li><li>通过使用先前描述的Western印迹方法转移通过SDS-PAGE分离到PVDF膜上的蛋白质。17,24,27-31 <ol><li>方框用5％无脂奶在膜上的非特异性结合位点在1×PBS中，然后孵育稀释的初级抗体的溶液的膜在RT 1小时。请参阅各主要抗体的详细信息（ 例如，稀释比例）材料/设备清单。 </li><li>用PBS-T缓冲液洗涤该膜3-4次（每3到5分钟孵育振荡）后（1×PBS和0.1％吐温-20），孵育在近红外（IR）的混合物，该膜荧光染料缀合的二级抗体（第1稀释比:20,000），DyLight缀合的第二抗体（1稀释比:20,000），和/或辣根过氧化物酶（HRP）缀合的二级抗体（在PBS-T稀释; diluti上的比例为1:在RT 1小时10,000）。请参阅各二级抗体的详细信息（ 例如，稀释比例）材料/设备清单。 </li></ol></li><li>洗膜3次，然后扫描该膜，以分析与红外成像系统和/或用于免疫印迹检测的化学发光成像的蛋白质（参见材料/设备的列表）。 <ol><li>对于使用红外成像系统，请参阅（6.4.1）的补充代码文件。 </li><li>对于使用化学发光成像仪，见（6.4.2）的补充代码文件。使用超灵敏化学发光增强（ECL）底物（见材料/设备清单）这个系统。 </li></ol></li></ol>

作者宣称，他们没有竞争的经济利益。

近年来，许多研究已经在着丝粒动粒生物学为固定细胞开发了不同的定量显微镜测定。42进展通常需要的蛋白质的着丝粒特异性或特异性着丝点功能的理解，其中亚细胞时空调控反映不断变化的功能在细胞周期的这些蛋白质。因此，这里我们开发IIF染色和定量的IIF分析特异性分析内源性和外源性CENP-A蛋白的相对水平，这可以适用不同处理的样品中的方法。目前，我们实现了内源性CENP-I，CEN...

"着丝粒"被经典定义为遗传学抑制减数分裂重组的区域和以后确认为有丝分裂染色体的主收缩，其中有丝分裂期间起着准确的染色体分离中起重要作用。 "着丝粒"被描述为结合到微管在着丝粒的表面上，所揭示的电子显微镜的多层结构; "着丝粒"后来被定义为在本地化有丝分裂的着丝粒的大分子复合物。尽管脊椎动物中的着丝粒DNA序列的戏剧性发散，动粒的结构和组成是高度保守的。需要有丝分裂过程中染色体分离友们纺锤体微管和动粒之间的动态互动，在着丝粒动粒功能导致非整倍体，从而癌症的缺陷。 在大多数真核生物着丝粒没有定义的DNA序列，而是由包括171 bp的α卫星DNA分子的重复DNA白斑样的大阵列（0.3-5兆）的。除了 ​​在芽殖酵母，着丝粒的身份不是由DNA序列，但由包含组蛋白H3变体CENH3特殊核小体存在下实现（着丝粒蛋白A [CENP-A〕在人类中）。5 CENP-A的核小体定位于哺乳动物着丝粒7并绑定到171 bp的α卫星DNA分子的内板。主动要求着丝粒CENP-含A-核直接组成着丝粒​​相关网络（CCAN）和动粒蛋白的募集，它们共同规范染色体通过纺锤体检查点的有丝分裂纺锤体和随后的周期进程的附件。 另外，在上述证据的光，CENP-A已被提出是着丝粒8的后生标记;然而，这个过程由CENP-A结合我n要着丝粒DNA并负责此掺入的因素尚未充分表征。短丝粒靶向域（CATD）驻留在组蛋白折叠CENP-A的区域和替换H3与CATD相应区域的是足以直接H3到着丝粒。9几项研究建议的翻译后的功能角色修改CENP-A 12-16（PTM）;然而，CENP-A的招募这些翻译后修饰的至着丝粒的分子机制尚未阐明。我们以前报道，CUL4A-RBX1-COPS8 E3连接酶的活性需要CENP-A到着丝粒CENP-A K124泛素化和本地化。17 着丝粒蛋白的发现和鉴定，导致对染色体分离的新见解。18 100多名动粒的成分已通过各种途径在脊椎动物细胞中鉴定。19,20的下在荧光显微镜的着丝点是如何组装和功能也来自各着丝粒动粒蛋白和蛋白质网络的细胞功能的细胞内的表征地位。19直接可视化和先进的成像方法提供着丝粒着丝粒组件非凡分辨率及允许直接观察着丝粒和着丝粒的特定的分子组件。此外，使用特异性抗体间接免疫荧光的方法（IIF）染色对这些观察结果是至关重要的。然而，尽管关于IIF协议，很少在着丝粒特异性着丝粒蛋白质的细节问题的讨论的许多报告。1-4。因此，显影和报告IIF染色和定量IIF测定方法具体分析每个着丝粒动粒蛋白是极其重要的。在IIF染色，应该与染色方案进行，以避免所感兴趣的蛋白的损失或单元的其余部分。然而，定影破坏抗原位点偶尔，以及不同的抗体-抗原组合与一种固定剂不佳工作，但很好地与另一种，21和固定剂的选择在很大程度上取决于所感兴趣的蛋白（多个）。因此，不同的固定剂的方法是在着丝粒动粒蛋白IIF染色至关重要。 间接免疫荧光（IIF）染色和测定以解​​决内源性着丝粒动粒蛋白质，包括CENP-A和Flag标记的外源性​​CENP-A蛋白，以及在人类细胞中这些蛋白质的定量本地化这里优化方法已被开发出来。这些方法可以应用到在其它物种的着丝粒，动粒蛋白质的分析。

<table><tbody><tr><td>Lipofectamin 2000</td><td>Life Technologies/Invitrogen</td><td>11668</td><td>transfection reagent I</td></tr><tr><td>Lipofectamin RNAiMAX</td><td>Life Technologies/Invitrogen</td><td>13778</td><td>transfection reagent II</td></tr><tr><td>Opti-MEM I</td><td>Life Technologies/Invitrogen</td><td>31985</td><td>Reduced serum media, warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>High-glucose DMEM (Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium)</td><td>Life Technologies/BioWhittaker</td><td>12-604</td><td>high-glucose DMEM, warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td><td>Life Technologies/Gibco</td><td>10082</td><td>FBS (fetal bovine serum)</td></tr><tr><td>Poly-L-Lysine SOLUTION</td><td>SIGMA-SLDRICH</td><td>P 8920</td><td>Poly-L-Lysine, 0.1% w/v, in water</td></tr><tr><td>UltraPure Distilled Water</td><td>Life Technologies/Invitrogen/Gibco</td><td>10977</td><td>Sterile tissue culture grade water&amp;nbsp;</td></tr><tr><td>Micro Cover glass (22 mm x 22 mm)&amp;nbsp;</td><td>Surgipath</td><td>105</td><td>Cover glass (22 mm x 22 mm)&amp;nbsp;</td></tr><tr><td>6 Well Cell Culture Cluster</td><td>Fisher/Corning Incorporated</td><td>07-200-83</td><td>6-well polystyrene plate&amp;nbsp;</td></tr><tr><td>Penicillin, Streptomycin; Liquid</td><td>Fisher/Gibco</td><td>15-140</td><td>Penicillin-streptomycin</td></tr><tr><td>PAP PEN&amp;nbsp;</td><td>Binding Site</td><td>AD100.1</td><td>Hydrophobic barrier pen (for a water repellant barrier in immunofluorescent staining)</td></tr><tr><td>Paclitaxel (Taxol)</td><td>SIGMA-SLDRICH</td><td>T7402</td><td>Taxol for mitotic cell analysis</td></tr><tr><td>TN-16, microtubule inhibitor (TN16)</td><td>Enzo Life Sciences</td><td>BML-T120</td><td>TN16 for mitotic cell analysis</td></tr><tr><td>BSA (bovine serum albumin)</td><td>SIGMA-SLDRICH</td><td>A7906</td><td>Blocking reagent</td></tr><tr><td>Triton X-100</td><td>SIGMA-SLDRICH</td><td>T8787</td><td>Detergent for permeabilization</td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-SLDRICH</td><td>P6148</td><td>Fixation reagant</td></tr><tr><td>DAPI</td><td>SIGMA-SLDRICH</td><td>D9542</td><td>For nuclear staining</td></tr><tr><td>p-phenylenediamine</td><td>SIGMA-SLDRICH</td><td>P6001</td><td>For mounting medium</td></tr><tr><td>VWR Micro Slides, Frosted</td><td>VWR International</td><td>48312-013</td><td>Micro slides&amp;nbsp;</td></tr><tr><td>Anti-CENP-A antibody</td><td>Stressgen/Enzo Life Sciences</td><td>KAM-CC006</td><td>Mouse monoclonal antibody; dilution ratio of 1:100 (IIF), 1:5000 (WB)</td></tr><tr><td>Anti-CENP-B antibody</td><td>Novus Biologicals</td><td>H00001059-B01P</td><td>Mouse monoclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation)</td></tr><tr><td>Anti-CENP-B antibody&amp;nbsp;</td><td>abcam</td><td>ab25734</td><td>Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation)</td></tr><tr><td>Anti-centromere antibody (ACA)</td><td>Fitzgerald Industries International, Inc.</td><td>90C-CS1058</td><td>Human centromere antiserum; dilution ratio of 1:2000 (IIF)</td></tr><tr><td>Anti-CENP-H antibody</td><td>Bethyl Laboratories</td><td>BL1112 (A400-007A)</td><td>Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF)</td></tr><tr><td>Anti-CENP-H antibody</td><td>BD</td><td>612142</td><td>Mouse monoclonal antibody; dilution ratio of 1:200 (IIF)</td></tr><tr><td>Anti-CENP-I antibody</td><td>N/A, Dr. Katusmi Kitagawa</td><td>N/A, Dr. Katusmi Kitagawa</td><td>Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF); Niikura et al., Oncogene, 4133-4146 (2006)</td></tr><tr><td>Anti-KNL1 antibody</td><td>Novus Biologicals</td><td>NBP1-89223</td><td>Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)</td></tr><tr><td>Anti-Hec1 antibody</td><td>Novus Biologicals / GeneTex</td><td>NB 100-338 / GTX70268</td><td>Mouse monoclonal antibody; dilution ratio of 1:100 (IIF)</td></tr><tr><td>Anti-Hec1 antibody</td><td>GeneTex</td><td>GTX110735</td><td>Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)</td></tr><tr><td>Anti-Ska1 antibody</td><td>abcam</td><td>ab46826</td><td>Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF)</td></tr><tr><td>Anti-Flag antibody</td><td>SIGMA-ALDRICH</td><td>F3165</td><td>Mouse monoclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB)</td></tr><tr><td>Anti-Flag antibody</td><td>SIGMA-ALDRICH</td><td>F7425</td><td>Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB)</td></tr><tr><td>Anti-CUL4A antibody</td><td>N/A, Dr. Pradip Raychaudhuri</td><td>N/A, Dr. Pradip Raychaudhuri</td><td>Rabbit polyclonal antibody; dilution ratio of 1:3000 (WB); Shiyanov et al., The Journal of biological chemistry, 35309-35312 (1999)</td></tr><tr><td>Anti-RBX1 antibody</td><td>Cell Signaling</td><td>4397</td><td>Rabbit polyclonal antibody; dilution ratio of 1:2000 (WB)</td></tr><tr><td>Anti-GAPDH antibody</td><td>Chemicon</td><td>MAB374</td><td>Mouse monoclonal antibody; dilution ratio of 1:5000 (WB)</td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG</td><td>Life Technologies/Invitrogen</td><td>A11001</td><td>fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)</td></tr><tr><td>Alexa Fluor 594 Goat Anti-Mouse IgG</td><td>Life Technologies/Invitrogen</td><td>A11005</td><td>fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)</td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit IgG</td><td>Life Technologies/Invitrogen</td><td>A11008</td><td>fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)</td></tr><tr><td>Alexa Fluor 594 Goat Anti-Rabbit IgG</td><td>Life Technologies/Invitrogen</td><td>A11012</td><td>fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)</td></tr><tr><td>Non fat powdered milk (approved substitution for carnation powdered milk)</td><td>Fisher Scientific</td><td>NC9255871 (Reorder No. 190915; Lot# 90629)</td><td>Skim milk</td></tr><tr><td>Leica DM IRE2 motorized fluorescence microscope&amp;nbsp;</td><td>Leica</td><td></td><td>motorized fluorescence microscope&amp;nbsp;</td></tr><tr><td>HCX PL APO 63x oil immersion lens</td><td>Leica</td><td>LEICA HCX PL APO NA 1.40 OIL PH 3 CS</td><td>63X oil immersion lens</td></tr><tr><td>HCX PL APO 100x oil immersion lens</td><td>Leica</td><td>LEICA HCX PL APO NA 1.40 OIL PHE</td><td>100X oil immersion lens</td></tr><tr><td>Leica EL6000 compact light source</td><td>Leica</td><td></td><td>External compact light source for fluorescent excitation</td></tr><tr><td>ORCA-R2 Digital CCD camera&amp;nbsp;</td><td>Hamamatsu</td><td>C10600-10B</td><td>digital CCD camera&amp;nbsp;</td></tr><tr><td>Openlab version 5.5.2 Scientific Imaging Software&amp;nbsp;</td><td>Perkin Elmer/Improvision</td><td></td><td>For image observation, acquisition, quantification, and analysis</td></tr><tr><td>Velocity version 6.1.1 3D Image Analysis Software&amp;nbsp;</td><td>Perkin Elmer/Improvision</td><td></td><td>For image observation, acquisition, quantification, and analysis</td></tr><tr><td>Complete EDTA-free protease inhibitor cocktail</td><td>Roche</td><td>11873580001/11836170001</td><td>Protease inhibitor cocktail tablets</td></tr><tr><td>PlusOne 2-D Quant Kit</td><td>Amersham Biosciences</td><td>80-6483-56</td><td>Commercial protein assay reagent I for measurement of protein concentration (compatible with 2% SDS)</td></tr><tr><td>Bio-Rad Protein Assay</td><td>Bio-Rad</td><td>500-0006</td><td>Commercial protein assay reagent II for measurement of protein concentration (compatible with 0.1% SDS)</td></tr><tr><td>Immobilon-FL</td><td>EMD Millipore</td><td>IPFL00010</td><td>PVDF membrane for transferring</td></tr><tr><td>IRDye 800CW Goat Anti-Mouse IgG</td><td>LI-COR Biosciences</td><td>926-32210</td><td>IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)</td></tr><tr><td>IRDye 680 Goat Anti-Rabbit IgG</td><td>LI-COR Biosciences</td><td>926-32221</td><td>IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)</td></tr><tr><td>Goat anti-Mouse IgG DyLight 549</td><td>Fisher Scientific</td><td>PI35507</td><td>DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)</td></tr><tr><td>Goat anti-Rabbit DyLight 649</td><td>Fisher Scientific</td><td>PI35565</td><td>DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF)</td></tr><tr><td>Goat anti-mouse IgG-HRP</td><td>Santa Cruz</td><td>SC-2005</td><td>HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF)</td></tr><tr><td>Goat anti-rabbit IgG-HRP</td><td>Santa Cruz</td><td>SC-2004</td><td>HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF)</td></tr><tr><td>Openlab version 5.5.2. Scientific Imaging Software&amp;nbsp;</td><td>Improvision/PerkinElmer</td><td></td><td>Software A</td></tr><tr><td>Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition)</td><td>PerkinElmer</td><td></td><td>Software B1</td></tr><tr><td>Volocity version 6.3 3D Image Analysis Software (Volocity Quantification)</td><td>PerkinElmer</td><td></td><td>Software B2</td></tr><tr><td>Branson SONIFIER 450</td><td></td><td></td><td>Sonicator</td></tr><tr><td>Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm</td><td>VWR Scientific Products Inc.</td><td>&amp;nbsp;33995-325</td><td>Disruptor horn for sonication</td></tr><tr><td>Branson Ultrasonics sonicator Microtip Tapered 6.5 mm</td><td>VWR Scientific Products Inc.</td><td>&amp;nbsp;33996-185</td><td>Microtip for sonication</td></tr><tr><td>Odyssey CLx Infrared imaging System&amp;nbsp;</td><td>LI-COR Biosciences</td><td></td><td>Infrared imaging system for immunoblot detection</td></tr><tr><td>Image Studio Analysis Software Ver 4.0&amp;nbsp;</td><td>LI-COR Biosciences</td><td></td><td>Software C</td></tr><tr><td>Molecular Imager Versadoc MP4000 System&amp;nbsp;</td><td>Bio-Rad</td><td></td><td>Chemiluminescence imager for immunoblot detection</td></tr><tr><td>Quantity One 1-D analysis software&amp;nbsp;</td><td>Bio-Rad</td><td></td><td>Software D</td></tr><tr><td>SuperSignal West Femto Maximum Sensitivity Substrate</td><td>Thermo</td><td>34095</td><td>Ultra-sensitive enhanced chemiluminescent (ECL) substrate</td></tr></tbody></table>

immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

内源性CENP-A的免疫荧光分析支持CUL4A-E3连接酶需要CENP-A的本地化着丝粒的假说 我们最近的研究表明，CUL4A-RBX1-COPS8 E3连接酶的活性需要在CENP-A和CENP-A至着丝粒的本地化赖氨酸124（K124）的泛素化17最初，相间着丝复合体（ICEN）被隔离抗CENP-A:原生染色质免疫沉淀，32-34，它一直推测，一些ICEN蛋白质可能在定位CENP-A至着丝粒的作用。因此，进行...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins at JoVE.com

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

immunofluorescence-analysis-endogenous-exogenous-centromere

Research

JoVE Journal

Biology

15.1K 次观看.  Nationwide Children's Hospital. Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

视频: Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

科研

遗传学

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

生物化学

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

评估小鼠卵母细胞中纺锤体组件检查点完整性

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

发育生物学

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

生物学

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

在飞睾丸成像中心体

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

衣藻中心粒单粒子重建的分离与荧光成像

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

摘要

探索更多视频

此视频中的章节

DNA 修饰的免疫: 共焦图像的计算分析

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

评估小鼠卵母细胞中纺锤体组件检查点完整性

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

在飞睾丸成像中心体

定量免疫检测，以衡量在中心体的变异蛋白水平

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

衣藻中心粒单粒子重建的分离与荧光成像

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A）的泛化

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

摘要

探索更多视频

此视频中的章节

DNA 修饰的免疫: 共焦图像的计算分析

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

评估小鼠卵母细胞中纺锤体组件检查点完整性

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

在飞睾丸成像中心体

定量免疫检测，以衡量在中心体的变异蛋白水平

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

衣藻中心粒单粒子重建的分离与荧光成像

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A）的泛化