Name: analysis of histone antibody specificity with peptide microarrays
Uploaded: 2017-08-01T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

这项工作得到Van Andel研究所的一部分支持，以及美国国家卫生研究院（CA181343）对SBR的研究资助

1.安装并运行ArrayNinja <ol><li>从www.virtualbox.org下载并安装Oracle Virtual Box。 </li><li>从http://research.vai.org/Tools/arrayninja下载并解压缩ArrayNinja虚拟机（VM）。 </li><li>打开虚拟框，并通过单击"机器"，"添加"添加ArrayNinja VM，然后从保存了ArrayNinja VM的文件夹中选择arrayninja.vbox。 </li><li>通过在Virtual Box中选择它并点击绿色的"开始"箭头启动ArrayNinja。 </li><li>虚拟框将打开一个新窗口，并显示一条消息，通过将Web浏览器导航到localhost可以访问VM:2080。60;注意:ArrayNinja的集装式版本也可通过 hub.docker.com/r/bradley.dickson/arrayninja/获取</li></ol> 2.设计阵列幻灯片和源板布局<ol><li>在ArrayNinja界面的"计划幻灯片布局"标题下，单击与正在使用的微阵列打印机对应的链接。 注意:ArrayNinja已被编程为模拟两种常用微阵列打印机的机器人运动（ 见表1）。可以根据要求配置与其他阵列的兼容性。 </li><li>单击"加载板"对话框中的"空"，然后单击"输入"。有关ArrayNinja设计模块的屏幕截图，请参见图2 。 </li><li>调整光斑直径为275μm，光斑间距为375μm。调整剩余的设置（板块/板排，总板排，复制，y中的特征，超级阵列，SuperA软糖;参见图2 ），以自定义功能如何在微阵列幻灯片上显示。 注意:光斑直径取决于微阵列针脚的大小。随着这些设置的调整，卡通幻灯片将实时更新。使用此卡通来预览每个设置如何修改最终的幻灯片布局。 </li><li>在幻灯片上的功能布局完成后，将鼠标悬停在每个唯一功能上，并在弹出的对话框中输入功能标识符。 注意:功能标识符可以由数字，字母或组合组成。这仅适用于唯一功能，当选择复制时，不会显示对话框。 </li><li>在所有唯一功能分配了一个标识符之后，点击"填充"。输入幻灯片布局的名称，然后点击"打印您的盘子"保存。将打开一个新页面，显示制作所选滑块设计所需的384孔源板数，以及映射要加载在源板中的每个要素的物理位置的表格。 注意:记住此名称，因为它将用于在分析微阵列数据时调用此设计（参见第6.2节）。点击"打印你的盘子"将布局保存在ArrayNinja中。 </li></ol><img alt="图2" class="xfigimg" src="/files/ftp_upload/55912/55912fig2.jpg" /> 图2:ArrayNinja设计模块。的屏幕截图<html> ArrayNinja设计模块以虚线显示。控制面板（上图）显示了可在微阵列打印机上更改的所有参数。随着这些参数的调整，幻灯片布局（左下角）的卡通图像实时更新。设置布局后，用户可以将鼠标悬停在各个点上，以输入唯一的功能标识符。来自该用户的ArrayNinja结构输入制作指定微阵列幻灯片布局所需的源平板（右下）中每个要素的位置的映射。 <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 3.制造微阵列<ol><li>准备源板<ol><li>使用第2.5节中使用ArrayNinja生成的地图创建384孔源板。 注意:在这个平台上查询的肽的详细描述可以在别处找到s ="xref"&gt; 17。 </li><li>将每个特征（ 例如 ，生物素化的组蛋白肽）1-2μL沉积到384孔源板的正确孔中。 注意:生物素化的组蛋白肽通常从200-400μM储备溶液中沉积，这等于在单个阵列斑点中10至25倍摩尔过量的肽与链霉亲和素结合位点。这是使用以下公式计算的: <img alt="方程" src="/files/ftp_upload/55912/55912eq1.jpg" /> 其中V是由销引起的体积，[ P ]是要打印的特征的浓度， N A是Avogadro的数量， S是点的表面积。 C是载玻片的覆盖率，表示为每单位面积的链霉亲和素分子数乘以3（可用的链霉亲和素结合位点的平均数）。 V和C由相应的制造商获得。其他功能可能需要不同的浓度，这取决于生物分子的大小，其中拥挤可能是一个问题。应经验测试每种新型特征的一系列印刷浓度，以确定最佳印刷浓度。 </li><li>将每个特征稀释10倍，并加入补充有1％牛血清白蛋白（BSA）的1x印刷缓冲液。在室温下将源极板以500×g旋转2分钟。 注意:推荐在印刷缓冲液中加入荧光素标记的生物素（5μg/μL）作为斑点控制，并作为视觉辅助，以便于分析过程中正确的阵列对准（ 见图4 ）。 </li></ol></li><li>打印协议（ 图3A -B ）。 <ol><li>通过清空废物收集容器并用无菌蒸馏水填充洗涤液容器和加湿器容器来准备排液器。 </li><li>输入参数used将ArrayNinja（第2节和图2 ）中的幻灯片设计到微阵列打印机控制程序中。 </li><li>使用微阵列打印机控制程序，设置1次洗涤的洗涤步骤。设置后清洗设置，每次清洗后重新浸渍5次。将湿度设置为60％。 注意:最佳洗涤配置可能因使用的微阵列打印机而异。根据所使用的微阵列打印机，最佳的洗后针浸渍配置可以变化。 </li><li>将功能化的载玻片（ 例如 ，链霉抗生物素蛋白包被的玻璃）插入基板压板，并将所有的压板放入压板升降机。将源板插入板架，并放入源板升降机。 </li><li>点击"打印"。监控打印过程中的几轮特征沉积，以确保所有的洗涤和浸渍设置是正确的。打印运行完成后，删除衬底从阵列器压出。 注意:当大的打印运行禁止在一天内完成阻塞步骤时，印刷的幻灯片可以在4℃的加湿室中孵育过夜。将玻璃杯旁边的一个小烧杯放在用塑料包装密封的纸板箱内。 </li><li>在室温下用封闭缓冲液封闭载玻片30分钟混合。 </li><li>在室温下用磷酸盐缓冲盐水（PBS），pH 7.6混合洗涤载玻片2×10分钟。通过在微阵列载玻片离心机中在室温下旋转30秒来干燥载玻片。 注意:为了一次处理大量的载玻片，可以使用高通量显微镜载玻片清洗室，以便平行洗涤50个载玻片。 </li><li>对于设计为用蜡分隔的滑块，请参见4.1节。对于所有其他设计，请在4°C下保存不受光照和潮湿的幻灯片。 注:印刷生物素化组蛋白肽以这种方式存储至少6个月是稳定的。 </li></ol></li></ol> 4.分割微阵列幻灯片<ol><li>疏水蜡笔（ 图3C ） <ol><li>使用蜡笔在包含特征的区域上涂蜡。让蜡在空气中干燥5分钟，然后再进入第5部分。 注意:阻塞后，阵列斑点可能很难通过眼睛可视化。来自ArrayNinja的幻灯片设计可以打印成比例尺，并用作涂蜡的指南。 </li></ol></li><li>硅垫片（ 图3D ） <ol><li>将透明膜从阵列垫片的背面剥离，并将粘合剂侧面放在微阵列载片上。 </li><li>在进入第5部分之前，将垫圈固定到位。 </li></ol></li><li>蜡印记（ 图3E ） <ol><li>对于设计为用蜡分隔的载玻片，使用10％BSA在普通玻璃上打印测试载玻片。使用此测试滑块优化蜡打印机导轨或阵列设置，以确保所有功能都将在蜡模腔内。 </li><li>将微阵列蜡打磨机加热至85°C，直到所有蜡完全熔化，约30分钟。 </li><li>将打印面朝下插入幻灯片，将幻灯片一直推到微阵列打印机上的右侧导板上。拉动杠杆使模具与滑块的表面接触。持续2秒。 注意:可以更改保持时间以达到最佳蜡边界厚度。 </li><li>快速移除幻灯片，目视检查蜡边界，确保所有的井都被封闭，并且边界不那么厚，以致它们侵入斑点。在4°C保存幻灯片，防止潮湿和光线。 注意:可以增加或减少保持时间以获得更厚或更薄的边界。 </li></ol></li></ol><img alt ="图3"class ="xfigimg"src ="/ files / ftp_upload / 55912 / 55912fig3.jpg"/&gt; 图3:微阵列制造。 （A）使用接触式微阵列打印机的链霉抗生物素蛋白包被的显微镜载玻片上的组蛋白肽微阵列制备。 （B）使用48×48格肽肽特征的3个子阵列制造的微阵列。 （C） 3个亚基与疏水性蜡笔的分离， （D） 2个硅胶粘合剂的子阵列和（E）具有蜡印痕的48个子阵列。所示的所有微阵列使用25×75mm显微镜载玻片制造。 <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 5.将组蛋白PTM抗体与肽微阵列杂交<ol><li>准备杂交缓冲液（PBS，pH 7.6,5％BSA，0.1％Tween-20）。 </li><li>在载玻片杂交缓冲液中平衡使用杂交容器。在杂交缓冲液中完全覆盖整个载玻片，并在低速下在轨道振荡器上在4℃下孵育30分钟。 </li><li>在杂交缓冲液中制备含有稀释的组蛋白PTM抗体的溶液。 注意:在实施例数据中，组蛋白抗体＃1和抗体＃2在杂交缓冲液中以1:1000稀释。推荐与免疫印迹相似的稀释范围作为起点。 </li><li>在4℃下用抗体溶液孵育阵列1小时。去除抗体溶液，并在4℃用冷PBS，pH 7.6洗涤阵列3次，5分钟。 </li><li>在杂交缓冲液中制备1:5,000 - 1:10,000稀释荧光染料缀合的二抗。 </li><li>在4℃下用二抗溶液孵育阵列30分钟，防止光照。取出二次抗体溶液，并在4℃用PBS，pH 7.6洗涤微阵列载玻片3次5分钟。蘸将微阵列置于含有0.1x PBS，pH 7.6的50mL锥形管中，以在室温下除去过量的盐。在微阵列载玻片离心机中室温干燥。 </li><li>按照微阵列扫描仪制造商推荐的扫描方案，用微阵列扫描仪扫描幻灯片，分辨率为25μm或更高。 注意:如果存在荧光素标记的生物素示踪剂，扫描绿色通道（例如:488 nm，em:509 nm）和对应于荧光染料缀合的二级抗体的通道，通常为红色（例如:635 nm，em :677nm）。扫描的目的是获得可以合并到单个.png文件中的单通道.tif文件（如第6.1节所述）。 </li></ol> 6.使用ArrayNinja分析微阵列数据<ol><li>准备合并的微阵列图像 注意:本节的目标是创建一个.png映像文件，它合并两个单通道.tif文件（在第5节中获得）。这个是与ArrayNinja分析模块兼容的唯一图像格式。以下说明代表了一种可能的方法来获取合并的图像文件。然而，其他解决方案可用（ 例如 ，免费的ImageJ）。 <ol><li>从安装了免费软件ImageMagick的计算机的bash终端中的命令行，导航到包含单个通道.tif文件的文件夹，并复制/粘贴以下步骤，在每个步骤之间进入"enter"（6.1.4 - 6.1 0.7）。 注意:大写字母的文件名称（ 例如 ，RED_CHANNEL）应替换为微阵列幻灯片图像的文件名。 </li><li>如有必要，首先使用命令&#39;convert INPUT.tif -negate OUTPUT.tif&#39;来反转图像。如果扫描器以白色信号保存.tif文件，并以黑色背景保存，则这是必需的。 <ol><li> convert -depth 16 RED_CHANNEL.TIF -clone 0-channel GB -evaluate set 0 -delete 0 out.png 2&gt; error.file。 </li><li> CONVert - depth 16 CONTROL_CHANNEL.TIF-克隆0通道RB -evaluate set 0 -delete 0 outa.png 2&gt; error.file。 </li><li> convert -depth 16 CONTROL_CHANNEL.TIF -clone 0-channel RG -evaluate set 0 -delete 0 outB.png 2&gt; error.file。 </li><li> convert out.png outa.png outB.png -set colorspace RGV -combine merged.png。 注意:名为"merged.png"的文件将保存在与原始.tif文件相同的文件夹中。 </li></ol></li></ol></li><li>使用ArrayNinja量化数据<ol><li>打开ArrayNinja，然后点击"量化具有已知源板的图像"下的相应微阵列打印机链接。在"装载板"对话框中输入保存的幻灯片设计的名称（步骤2.5），然后单击"进入"。 ArrayNinja分析模块的截图见图4 。 </li><li>单击"选择文件"并导航到并选择第6.1节中创建的"merged.png"文件。合并的影像e将载入以及幻灯片布局的网格。 </li><li>使用ArrayNinja控制面板底部的滑块调整对比度，亮度和中点。 注意:这些调整仅用于可视化目的，对定量没有影响。 </li><li>选择"分辨率"并输入与扫描图像的分辨率相匹配的值。使用"边缘"和"边缘顶部"将网格移动到与阵列上的点对齐。根据需要进行调整，使网格尽可能接近每个点。 </li><li>点击"Spot Seek"，然后等到按钮返回到原始的灰色。 注意:这可以重复几次，以将网格圆圈居中在各个点上。寻求功能使网格朝向每个点放松，以微调对齐。 </li><li>将"超级数组"值更改为"1"以单独处理每个子数组面板（如果需要）。如果使用4x 12蜡印刷幻灯片布局（ 图3E ），使用"iPin""jPin"和"subA"控件关闭功能来分析任何所需的4×12孔组合。例如，为了分析左上和右上孔的重复，将iPin中输入"1 4"，将"1 1"输入"1"，将"1 4"输入"子"。按&#39;enter&#39;。 </li><li>将鼠标悬停在各个位置以查看该功能的身份。 </li><li>点击"切换缩放"更仔细地检查功能。当鼠标在一个点上时，按"R"键选择背景参考点。参考点将以橙色突出显示。 注意:在"切换缩放"模式下，鼠标悬停的功能的放大图像显示在右上角。的额外的背景校正特征在ArrayNinja的详细讨论在别处讨论28。 </li><li>通过在将鼠标悬停在受影响的位置上的同时按"A"键，切断斑点（具有广泛变化的斑点形态或碎片，影响准确定量）。灭活点将变白。 </li><li>点击"填充"，然后点击"提交"来量化点数。 注意:一个新标签将打开并显示标准化为最亮斑点平均数据的条形图。条形图下面的表格包含归一化数据值和原始数据值。该表可以复制到电子表格中进行归档和进一步分析。 </li></ol></li></ol><img alt="图4" class="xfigimg" src="/files/ftp_upload/55912/55912fig4.jpg" /> 图4:ArrayNinja分析模块。显示ArrayNinja分析模块的屏幕截图。控制面板（左上角）显示了可调整的所有参数，以便可视化阵列，找到斑点，并对齐一个网格阵列图像。将鼠标悬停在功能上可显示放大视图（右上角），并显示包含与该功能相关联的识别信息（底部）的弹出窗口。用于背景校正的参考点为橙色。从下游分析中排除的特征是白色的。 ArrayNinja包含基于文本的搜索功能，以黄色突出显示匹配的功能，如H4K16的示例所示。 <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

作者没有什么可以披露的。

在生物医学研究中的应用抗体可靠性是最重要的46，47。在染色质生物化学中尤其如此，鉴于抗体的位置是开发用于表征组蛋白PTM的丰度和分布的大多数技术的关键工具。这里提出的方案详细介绍了用于分析组蛋白PTM抗体特异性的肽微阵列的设计，制造和使用的优化管线。该管道已被用于筛选大量市售和广泛使用的组蛋白PTM抗体，并且通过在线和扩展的组蛋白抗体特异性数据库（www.histoneantibodies.com） 17免费获得这些实验产生的数据。 从我们和其他人的工作来看，常见的情况是不利的组蛋白PTM抗体行为会使解释复杂化与这些试剂12，15，17生成的数据的离子。因此，有必要对抗体公司进行更严格和全面的质量控制措施。当选择试剂用于研究时，实验者和表观基因组联盟领导者（ 例如 ，ENCODE，BLUEPRINT）也需要自己评价组蛋白PTM抗体。此外，尽量减少批次间变异性，在表基因组映射平台上标准化使用高度验证的亲和试剂，并开发替代亲和力工具都是解决这一研究问题的可行的项目。 微阵列比基于微孔板的测定（ 例如 ELISA）具有许多优点，使得它们特别用于表征组蛋白PTM抗体特异性。微阵列能够平行和竞争性地分析数千个单独的肽 -抗体相互作用与最少的材料消耗在本文所述的方案中，每个肽特征的700皮摩尔足以产生100个微阵列载玻片。另外，可以使用少于1μg的抗体完成抗体分析，并且定制阵列格式可以使多个抗体和各种抗体稀释液平行筛选。 在微阵列上设计自定义功能布局可能是一项艰巨的任务。另外，每个新的微阵列设计都需要建立一个新的分析模板。我们发现这是实现微阵列技术的全面实用性的禁止。这促使我们ArrayNinja，互动，开放源码软件应用无缝集成的微阵列实验28的设计和分析方面的发展。 ArrayNinja的设计模块允许用户交互地设计幻灯片以适应实验所需的布局。 ArrayNinja virt通过映射打印机的机器人运动，将其转换为给定幻灯片设计的源板布局（ 图2 ）。创建自定义阵列格式现在是一个快速和常规的做法。 ArrayNinja的另一个关键特征是微阵列工作的设计和分析步骤之间的联系。使用设计模块中的用户定义的设置，ArrayNinja会叠加扫描的微阵列图像上的交互式网格，允许用户将鼠标悬停在任何功能上以显示其身份或搜索感兴趣的特征（ 图4 ）。 这个管道的几个关键步骤值得注意。首先，在设计打印布局时，应考虑包括足够的复制点，以便在数据收集中具有重要意义。另外，为了解决引脚对引脚的变化，每个特性都应该由至少两个不同的引脚进行存储。最后，也许最关键的一步是物理生物源极的离子。成功使用本协议中描述的高密度微阵列依赖于将每个特征的身份映射到最终幻灯片上的物理位置的能力。这个映射任务并不是微不足道的，但ArrayNinja通过提供一个平板映射大大地促进了这一步。在创建源板时，与该地图的任何偏差将导致分析期间的错误结论。 重要的是考虑使用微阵列来分析抗体特异性的局限性。而在阵列上捕获的脱靶组蛋白PTM抗体性质已被证实转化为具有相似的杂交条件的实验方法（ 例如 ，免疫印迹）17，18，在何种程度上抗体特异性的基于阵列的仿形转换芯片协议是暗示14,17而是华越来越关键的评价。 变化，以这条管道已经为组蛋白的读者，作家和橡皮擦23，24的分析在前面描述。修改这个平台的功能超出表观遗传学研究可以轻松适应任何可打印的感兴趣的图书馆。也可能使用比肽更复杂的材料构建用于染色质生物化学研究的微阵列。例如，现在在实验室设置48中常规生成显示各种PTM的重组核小体，并且在该生理学相关染色质亚基的上下文中分析组蛋白PTM抗体的特异性将是未来研究的一个令人兴奋的领域。 这里描述的方案的广泛使用的替代方法利用SPOT阵列技术，其中数百个肽在纤维素上直接合成失去膜支撑49 。然后膜本身可用于微阵列应用50 。从图书馆的复杂性和综合成本的角度来说，SPOT技术是有利的。然而，肽的使用SPOT方法合成的纯度已报道变化，并且共同的固相肽合成（ 例如 ，HPLC和质谱法）的质量控制和净化措施并不常规进行的50，51。另外，硝酸纤维素上的肽呈现不均匀，可以屏蔽表位。值得注意的是，两种类型的组蛋白肽阵列平台都可以商业用于无法获得合成肽和制备阵列所需的专门设备的用户。

基因组DNA通过组蛋白蛋白质优雅地包装在真核细胞核内，形成染色质。染色质的重复亚基是核小体，它由围绕组蛋白的八聚体核心卷绕DNA的147个碱基对- H2A，H2B，H3和H4 1。染色质广泛地组织成松散包装的真空染色质和紧密堆积的异染色质结构域。染色质紧密程度调节了蛋白质机制能够访问底层DNA进行基本DNA模板过程（如复制，转录和修复）的程度。 在染色质的上下文中的基因组可访问的关键调节剂是对组蛋白2，3的非结构化尾和核心结构域翻译后修饰。组蛋白PTMs通过影响染色质4的结构并间接作用直接起作用h招聘读者蛋白质及其相关的大分子复合物，其具有染色质重塑，酶促和脚手架活动5 。组蛋白PTM功能在过去二十年的研究绝大多数都表明这些标记在调节细胞命运，生物体发育和疾病发生/进展中起关键作用。由于基于质谱的蛋白质组学技术的进步，已发现超过20种独特的组蛋白PTM超过80种不同的组蛋白残基6 。值得注意的是，这些修改往往发生在组合，并与"组蛋白密码"假设是一致的，大量的研究表明，读者蛋白质通过识别组蛋白PTM的7，8，9的特定组合的有针对性的染色质的离散区域。向前迈进的一个关键挑战将是将功能分配给gr组蛋白PTM的不完整列表，并确定组蛋白PTM的具体组合如何协调与染色质相关的动态功能。 抗体是用于检测组蛋白PTM的lynchpin试剂。因此，已经商业开发了用于染色质生物化学研究的超过1,000种组蛋白PTM特异性抗体。随着高通量DNA测序技术的快速发展，这些试剂被ChIP-seq（染色质免疫沉淀加上下一代测序）的个别研究者和大规模表观遗传学"路线图"计划（ 例如 ENCODE和BLUEPRINT）广泛使用）等的管道以产生组蛋白PTM分发的全基因组10，11的高分辨率空间地图。然而，最近的研究已经表明，组蛋白PTM抗体的特异性可能是高度可变的，并且这些试剂显示不合适<html> avorable性能如脱靶表位识别，由相邻的PTM强阳性和负面影响，以及难以在特定残基鉴别修饰顺序（ 例如 ，单- ，二- ，或三甲基赖氨酸）12，13，14，15 ，16，17，18。因此，组蛋白PTM特异性抗体试剂的严格质量控制对于准确解释用这些有价值的试剂产生的数据是必要的。 微阵列技术能够以高通量，可重现和小型化的格式同时询问成千上万的大分子相互作用。为此，已经创建了各种微阵列平台来分析蛋白质-DNA 19 ，"&gt; 20，蛋白质21，和蛋白质-肽相互作用22。事实上，组蛋白肽微阵列已经成为一个信息发现平台染色质生物化学研究，使作家，橡皮擦的高通量分析，和翻译后修饰15组蛋白的读者，23，24，以及用于组蛋白抗体特异性的分析17，25。除了其在染色质和表观遗传学研究中的应用，组蛋白肽阵列具有潜在效用作为诊断/预后测试系统性红斑狼疮等自身免疫性疾病，其中抗染色质自身抗体产生26,27。 在这里，我们描述了我们为设计，制造和排队开发的集成管道使组蛋白肽微阵列产生识别组蛋白及其PTMs的抗体的特异性谱。我们最近开发的ArrayNinja是一个开源的交互式软件应用程序，其中集成了微阵列实验的设计和分析阶段28 。 ArrayNinja在Google Chrome中效果最好。简言之，机器人接触式微阵列打印机用于在链霉亲和素包被的玻璃显微镜载片上的限定位置沉积生物素 - 缀合的组蛋白文库。然后可以以竞争性和平行的测定形式使用数组来询问抗体 - 表位相互作用（ 图1 ）。肽文库由数百种独特的合成肽组成，分别含有PTM（赖氨酸乙酰化，赖氨酸/精氨酸甲基化和丝氨酸/苏氨酸磷酸化），以及大部分衍生自蛋白质组学数据集的相关组合。肽合成和验证方法<html>在其他地方详细23 。使用该阵列平台从我们正在进行的组蛋白PTM抗体筛选工作产生的数据归档在公共网络资源 - 组蛋白抗体特异性数据库（www.histoneantibodies.com）上。值得注意的是，与此协议的变型制造的组蛋白肽的微阵列也已被广泛地用于表征组蛋白PTM读者域8，29，30，31，32，33，34，35，36，37的活性以及最近个人资料组蛋白PTM作家和橡皮擦活动24 。 /files/ftp_upload/55912/55912fig1.jpg"/&gt; 图1:组蛋白肽微阵列上抗体筛选的逐步程序的动画描述。具有确定的翻译后修饰（红色和蓝色圆圈）的生物素化组蛋白肽与生物素荧光素在链霉抗生物素蛋白包被的玻璃上共同印刷。正相互作用可视化为红色荧光。 <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

<table><tbody><tr><td>Printing Buffer</td><td>ArrayIt</td><td>PPB</td><td></td></tr><tr><td>BSA</td><td>Omnipure</td><td>2390</td><td></td></tr><tr><td>Streptavidin-coated glass microscope slides</td><td>Greiner Bio-one</td><td>439003-25</td><td></td></tr><tr><td>polypropylene 384 well plate</td><td>Greiner Bio-one</td><td>784201</td><td></td></tr><tr><td>Biotin-fluorescein</td><td>Sigma</td><td>53608</td><td></td></tr><tr><td>contact microarray printer</td><td>Aushon</td><td>2470</td><td>Aushon 2470 Microarray Printer</td></tr><tr><td>contact microarray printer</td><td>Gene Machines</td><td>OmniGrid 100</td><td>OmniGrid Microarray Printer</td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190</td><td></td></tr><tr><td>Blocking Buffer</td><td>ArrayIt</td><td>SBB</td><td></td></tr><tr><td>Hydrophobic wax pen</td><td>Vector Labs</td><td>H-4000</td><td>ImmEdge Hydrophobic Barrier PAP Pen</td></tr><tr><td>Silicon Gasket</td><td>Grace Bio-labs</td><td>622511</td><td></td></tr><tr><td>Hybridization Vessel</td><td>Thermo Scientific</td><td>267061</td><td>or similar vessel</td></tr><tr><td>Fluorescent-dye conjugated secondary antibody</td><td>Life Technologies</td><td>A-21244</td><td>Alexa Fluor 647 (anti-rabbit)</td></tr><tr><td>Fluorescent-dye conjugated secondary antibody</td><td>Life Technologies</td><td>A-21235</td><td>Alexa Fluor 647 (anti-mouse)</td></tr><tr><td>Wax Imprinter</td><td>ArrayIt</td><td>MSI48</td><td></td></tr><tr><td>Tween-20</td><td>Omnipure</td><td>9490</td><td></td></tr><tr><td>Microarray Scanner</td><td>Innopsys</td><td>InnoScan 1100AL</td><td>or equivalent microarray scanner</td></tr><tr><td>EipTitan Histone Peptide Microarray</td><td>Epicypher</td><td>112001</td><td></td></tr><tr><td>AbSurance Pro Histone Peptide Microarray</td><td>Millipore</td><td>16668</td><td></td></tr><tr><td>MODified Histone Peptide Array</td><td>Active Motif</td><td>13001</td><td></td></tr><tr><td>Histone Code Peptide Microarrays</td><td>JPT</td><td>His_MA_01</td><td></td></tr><tr><td>Wax</td><td>Royal Oak</td><td>GulfWax</td><td>for wax imprinter</td></tr><tr><td>Humidified Microarray Slide Hybridization Chamber</td><td>VWR</td><td>97000-284</td><td></td></tr><tr><td>High throughput microscope slide washing chamber</td><td>ArrayIt</td><td>HTW</td><td></td></tr><tr><td>Microscope slide centrifuge</td><td>VWR</td><td>93000-204</td><td></td></tr><tr><td>Antibody 1</td><td>Abcam</td><td>8898</td><td></td></tr><tr><td>Antibody 2</td><td>Millipore</td><td>07-473</td><td></td></tr><tr><td>Biotinylated histone peptide</td><td>EpiCypher</td><td>12-0001</td><td>Example peptide. Similar peptides with various modifications are available from several commercial sources.</td></tr><tr><td>ImageMagick</td><td></td><td></td><td>https://www.imagemagick.org/script/index.php</td></tr><tr><td>ArrayNinja</td><td></td><td></td><td>https://rothbartlab.vai.org/tools/</td></tr></tbody></table>

analysis of histone antibody specificity with peptide microarrays

组蛋白蛋白的翻译后修饰（PTM）在调节染色质结构和基因表达方面的作用被广泛研究。组蛋白PTM特异性抗体的大规模生产和分布大大促进了对这些标记的研究。由于组蛋白PTM抗体是许多染色质生物化学应用的关键试剂，因此严格的抗体特异性分析对准确的数据解释和现场持续进展是必需的。该协议描述了用于分析组蛋白抗体特异性的肽微阵列的设计，制造和使用的集成管线。该方案的设计和分析方面由ArrayNinja（一种我们最近开发的开源和交互式软件包）来简化微阵列打印格式的定制。该管道已被用于筛选大量市售和广泛使用的组蛋白PTM抗体s，并且通过在线和扩展的组蛋白抗体特异性数据库免费获得从这些实验产生的数据。除组蛋白之外，本文描述的一般方法可广泛应用于PTM特异性抗体的分析。

该方案已被用于设计和制造用于分析组蛋白PTM抗体特异性的肽微阵列平台。该阵列查询了超过300个独特的肽特征（长度为20-40个残基）的文库，代表了核心和变体组蛋白38上发现的许多已知PTM组合。该管道已经成为筛选许多广泛使用和市售的组蛋白PTM抗体的主力，并且在组蛋白抗体特异性数据库（www.histoneantibodies.com）上提供了完整的数据集。 17 我们管道的主干是ArrayNinja，我们最近开发的开源软件应用程序，它集成了微阵列工作的规划和分析阶段28 。 ArrayNinja分析模块允许用户在信息中进行交互他们的原始阵列图像文件的方式。打印的特征标识自动与图像集成，通过将鼠标光标悬停在某个位置可以显示这些标识。这种集成还可以通过名称快速搜索感兴趣的特征，并从下游分析中排除特征。 ArrayNinja还提供了本地和非本地背景噪声校正的选项，以及适应斑点形态的校正方案。 ArrayNinja及其能力的全部细节可以在其他地方28被发现。 ArrayNinja计算每个肽特征的信号强度，并将这些强度聚合成基于特征标识的具有误差的平均值。返回原始和归一化的信号平均值，其中归一化常数由最亮特征平均值确定。每个肽的信号强度可用于比较相对亲和力<html>的抗体在微阵列的特征中。在这里，我们展示了用这种管道分析的两种抗体的代表性数据集，突出了在解释用这些试剂获得的结果时应考虑的表位识别特性（ 图5 ）。 脱靶识别是所有抗体的关注点，并且围绕可修饰的组蛋白残基的表位的共同作用使得组蛋白PTM抗体成为特别的挑战。实际上，提出识别组蛋白H3（H3K9me3）上的三甲基化赖氨酸9的抗体结合组蛋白H4（H4K20me3）上的H3K18，H3K27和赖氨酸20三羟甲基化的肽或者比H3K9me3更好的结合图 （ 图5A ）。围绕H3K9（ARKS）的序列与H3K27保守相关，组蛋白PTM抗体和染色质调节因子的交叉反应性在结合和修饰这些位点时已被注意到其他地方SS = "外部参照"&gt; 15，39，40，41。甲基赖氨酸抗体常见的另外观察到的脱靶识别性质是不能区分甲基顺序。这可以用与H3K4me2和H3K4me1肽交叉反应的H3K4me3抗体获得的结果来说明（ 图5B ）。甲基次序区分是重要的，因为研究已经表明，H3K4me3的，H3K4me2和H3K4me1被不同地在基因组和可能功能分布在不同的方式42，43。例如，H3K4me3位于最活跃转录基因的转录起始位点，而活性增强子通常被H3K4me1标记44 。 除了目标外识别，积极和消极的影响e是相邻PTMs的组蛋白抗体的常见观察性质。 图5A所示的H3K9me3抗体受邻近赖氨酸残基的乙酰基的积极影响。事实上，最近的质谱分析表明，乙酰基，特别是H3K14ac，经常与H3K9me3 38共存。 图5B所示的H3K4me3抗体由于苏氨酸6（H3T6p）的相邻磷酸化而受到负面影响。 H3T6p已知具有由读取器蛋白识别的H3K4me3的正面和负面影响，并且这可以作为一个动态机制来调节特定染色质的募集因子15，45。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55912/55912fig5.jpg" /> 图5:肽微阵列抗体分析。分析（A） H3K9me3（抗体＃1）和（B） H3K4me3（抗体＃2）抗体在肽微阵列上的分析。绿线/条表示仅含有目标PTM的肽。灰线/条表示使用单因素方差分析计算的信号强度与绿线/巴没有显着差异（p&gt; 0.05）的肽，其比较所有肽与靶肽的平均相对强度。红线和蓝线/条分别表示从绿线/条显着减少或增加的信号（p &lt;0.05）。橙色线条/条表示脱靶肽。数据显示为（左）在跨越H3和H4尾部的N-末端区域的肽的PTM复杂性的视觉图示，其中每行的宽度对应于针对该肽特征测量的相对强度，和（右）条形图从6个点测量显示相对信号强度平均值±SEM微阵列。本分析中包含的所有肽的长度为20个氨基酸，对应于组蛋白H3氨基酸1 - 20或15 - 34。 <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Analysis of Histone Antibody Specificity with Peptide Microarrays

用肽微阵列分析组蛋白抗体特异性

该手稿描述了将肽微阵列技术应用于识别组蛋白及其翻译后修饰的抗体的特异性分析的方法。

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

analysis-of-histone-antibody-specificity-with-peptide-microarrays

Nanyang Technological University

Research

JoVE Journal

Biochemistry

39.9K 次观看.  Van Andel Research Institute. 该手稿描述了将肽微阵列技术应用于识别组蛋白及其翻译后修饰的抗体的特异性分析的方法。 

视频: Analysis of Histone Antibody Specificity with Peptide Microarrays

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

科研

生物化学

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

免疫与感染

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

生物学

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

基于肽矩阵的 HBV 特异性 CD4 T 细胞响应和 HLA-DR 受限 CD4 T 细胞表位的识别分析

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

蛋白赖氨酸甲基转移使用SPOT肽阵列特异性分析

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

Source: Liu, P., et al. <a href="https://app.jove.com/v/56278">Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation.</a> J. Vis. Exp. (2017).
This video demonstrates an assay to detect anti-donor HLA alloantibodies in organ recipients. A peptide array is synthesized to represent potential epitopes for antibody-mediated organ rejection. The array is incubated with the recipient&#39;s serum, containing alloantibodies that bind to their target epitopes. Antibody binding is detected using chemiluminescence to identify donor-specific HLA epitopes that incite alloantibody reactivity.

器官受体中基于肽阵列的抗供体 HLA 同种抗体检测

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Design of Custom Array Layout and ...