我们感谢有益的讨论Tuschl实验室成员。氢是由德国学术Austauschdienst（DAAD）的支持。这项工作是支持由瑞士国家基金拨款＃3100A0 - 114001锰锌; TT是霍华德休斯医学研究所研究员，并在他的实验室的工作是由美国国立卫生研究院拨款GM073047和MH08442和斯塔尔基金会的支持。

该协议说明HEK293细胞表达的标志/标记后，用强力霉素诱导的HA - IGF2BP1的PAR -剪辑过程。我们将使用免疫抗旗的抗体。 PAR -剪辑将与任何细胞表达内源性的检测水平，未标记的RNA结合蛋白（RBP）的利益，如果一个高效的免疫抗体。 扩大细胞<ol><li>展开FlpIn-HEK293/TO/FLAG/HA-IGF2BP1细胞生长的培养基。我们建议使用100-400 x 10 6个细胞（约10-40 15厘米细胞培养板）之间为出发点。种植约80％汇合。 </li><li>交联前14 h 添加）4 thiouridine直接向细胞培养液中的终浓度为100微米（1米4 thiouridine原液1:1000 V / V） 和b）诱导表达的旗帜/医管局标签增加1微克/毫升强力霉素（10毫克/毫升强力霉素原液1:10,000 V / V）IGF2BP1。注意：你可以代替4 - thiouridine还可以使用6 thioguanosine 100微米。 </li></ol>紫外光交联<ol><li> 10毫升每盘冰冷的PBS洗涤细胞一次，并完全删除PBS。 </li><li>与0.15焦耳/平方厘米 365纳米的紫外线光在Stratalinker 2400（Stratagene公司）或类似的设备发现的冰和照射托盘板。 </li><li>刮取细胞，每1毫升PBS橡胶板，转移至50 ml离心管警察和500 XG在4 ° C 5分钟，离心收集上清液。 100 × 10 6 HEK293细胞（10 15厘米的板），将产生约。 1毫升的湿颗粒细胞。 </li><li> （可选），除非你想继续直接与细胞裂解，休克冻结在液态氮和存储细胞沉淀于-80 ° C。细胞沉淀，可存储至少12个月。 </li></ol>细胞裂解和消化RNaseT1 <ol><li>在3卷1X NP40裂解液交联细胞的颗粒细胞，并在冰上孵育10分钟。 </li><li>清除细胞裂解液通过离心13,000 15分钟XG在4 ° C。 </li><li>进一步清除裂解液过滤0.2微米膜注射器过滤器（PALL Acrodisc或同等学历）。 </li><li>核糖核酸酶T1（Fermentas公司，万U /μL）添加到终浓度为1 U /μL，在15分钟的水浴孵育在22 ° C。随后冰在出发前5分钟的冷却反应。 </li></ol>免疫沉淀和交联的目标RNA片段恢复利用磁选整个样品制备按照这些指引，以防止干燥磁珠。 <ol><li>将管内含有1 2分钟的磁立场上的珠子。 </li><li>添加缓冲管，而管磁选。 </li><li>第磁选管，删除，重悬珠。您可以重悬，用手指轻弹管珠或使用设置在5月6日vortexer。 </li><li>短暂离心收集任何可能留在管帽珠。 </li><li>重复步骤1到4，根据需要。 </li></ol>磁性微球的制备<ol><li>转移10μL磁珠G蛋白的磁性粒子（Invitrogen）的每毫升细胞裂解液（一个典型的实验应该约40 50μL珠）到1.5毫升离心管。柠檬酸磷酸盐缓冲液1毫升，两次清洗珠。 </li><li>悬浮在柠檬酸 - 磷酸盐缓冲液相对原始卷珠悬浮量的两倍。 </li><li>新增反旗M2单克隆抗体（Sigma公司）的0.25微克每毫升悬浮和旋转的轮子上在室温下40分钟的孵育。 </li><li> 1毫升柠檬酸磷酸盐缓冲液洗珠两次，以除去未结合的抗体。 </li><li>在柠檬酸 - 磷酸盐缓冲液相对原始卷珠悬浮量的两倍重悬珠。 </li></ol>免疫沉淀（IP），第二个核糖核酸酶T1消化，和去磷酸化<ol><li>添加20μL新鲜配制每毫升含部分核糖核酸酶T1处理过的细胞裂解液抗体偶联的磁珠孵育1小时旋转的轮子上在15毫升离心管4 ° C。 </li><li> 15日和50毫升离心管（Invitrogen）的磁珠上的磁性粒子收集器收集，并转移到1.5 ml离心管。 </li><li>洗净珠在1毫升IP缓冲液洗3次。 </li><li>添加RNaseT1（Fermentas公司，万U /μL）至终浓度为100 U /μL和珠悬浮在一个15分钟的水浴孵育22 ° C。随后对冰酷5分钟。 </li><li>洗净珠在1毫升高盐缓冲液洗3次。 </li><li> 1体积的去磷酸化缓冲液中的悬浮珠</li><li>小牛肠碱性磷酸酶（CIAP，NEB）添加到终浓度为0.5 U /μL，并暂停10分钟在37 ° C。 </li><li> 1毫升磷酸缓冲液洗两次洗珠</li><li>洗两次多聚核苷酸激酶（PNK）缓冲无DTT（酶反应所必需的数码地面电视浓度高到足以破坏磁珠）珠。 </li><li>在一个原珠PNK缓冲量悬浮珠</li></ol>放射性标记的交联免疫沉淀蛋白质的RNA片段<ol><li>上文所述珠悬浮，添加Υ- 32 P - ATP到0.5μCi/μL和T4 PNK的最终浓度（NEB）一个原珠卷1 U /μL 。孵育30分钟暂停在37 ° C。 </li><li>添加非放射性ATP获得终浓度为100μm，另有5分钟，在37 ° C。 </li><li> 800 PNK没有数码地面电视的缓冲液洗磁珠5倍。 </li><li>在70 SDS - PAGE上样缓冲液重悬珠。 </li></ol> SDS - PAGE和RNA -蛋白质复合物的交联电泳凝胶片<ol><li>孵育放射性标记的悬浮在加热块5分钟在95 ° C变性和交联的RNA和旋涡释放免疫沉淀RBP的。 </li><li>删除磁珠上的分隔，将上清转移到一个干净的1.5 ml离心管。 </li><li>负载40μL上清，每孔一个NOVEX双三4-12％（Invitrogen）的预制聚丙烯酰胺凝胶和凝胶运行55分钟，在200 V。 </li><li>拆开凝胶室和拆除凝胶，离开它装在一个盘子。为了方便phosphorimager纸打印输出凝胶对齐，我们建议三个微小的放射性凝胶片植入凝胶的四个角中的三个不对称。放射性凝胶片可以收集从以前用来净化放射性标记的合成寡核苷酸凝胶。包裹在塑料薄膜（如保鲜膜）的凝胶，以避免污染。 </li><li>凝胶暴露一个空白phosphorimager 1小时的屏幕和可视化上phosphorimager。 </li><li>将phosphorimager定向使用植入的凝胶块的打印输出上的凝胶。切出的条带，符合预期大小的RBP（IGF2BP1，约75 kDa的），并转移到一个D -管透析MIDI管，并添加800μL1X SDS的运行缓冲。 </li><li> Electroelute在1X SDS交联RNA - RBP的复杂运行在100 V 2小时的缓冲从凝胶切除和electroeluted 800μLSDS电泳缓冲液中的一个D -管透析MIDI（Novagen公司）根据制造商的指示。 </li></ol>蛋白酶K消化<ol><li>加入等体积的2倍蛋白酶K缓冲的electroeluate，蛋白酶K（罗氏）除了到终浓度为1.2毫克/毫升。孵育30分钟，在55 ° C。 </li><li>恢复酸性酚/氯仿/ IAA的提取（25:24:1，pH值4.0）由氯仿萃取的RNA。加入1μL的糖原（10毫克/毫升的股票）的RNA沉淀加入乙醇3卷。 10.5μl水溶解的沉淀。 </li></ol> cDNA文库的准备和深度测序回收的RNA进行通过一个标准的最初19监管的小分子RNA的克隆的cDNA文库制备协议。第一步，3&#39;适配器结扎，进行了使用10.5μL回收的RNA在20μL规模。使用公司Solexa测序适配器设置描述。根据回收的RNA量，5&#39; - 3&#39;适配器适配器没有插入的产品可作为额外的PCR带的cDNA扩增后检测。在这种情况下，消费预期大小较长的PCR产品从3％NuSieve低熔点琼脂糖凝胶，洗脱从胶件使用的GelElute试剂盒（Qiagen）和序列使用公司Solexa技术的PCR产物。一个公司Solexa测序运行通常给予读取一个转录的RNA结合蛋白的结合位点的覆盖面广，足够6和10万之间的序列。 生物信息学分析仔细序列的生物信息学分析的内容需要做的研究RBP的RNA结合位点，如RNA识别的元素，首选的限制性商业惯例的结合区域（外显子与内含子，获得有意义的见解，编码序列与非翻译序列）。需要对基因组和EST数据库对齐的序列读取。我们通常使用读取mappi吴独特的基因组与一个不匹配，插入或删除序列构建的集群读取，然后可以进一步分析。在集群测序的特征突变的频率读取，T到C转换采用​​4苏和G A转换时使用6 - SG的，成功交联的序列指示。根据我们的经验与4 -苏标记uncrosslinked RNA的一个背景的突变率约20％。这个比率是增加至约。后交联的50-80％。 一个生物信息学分析的详细描述，可以发现在该出版物的补充材料，哈夫纳等18。 可选步骤测定总RNA到4 thiouridine纳入各级从细胞株中分离的总RNA，稳定表达后，在100微米4SU 16小时收获前培养基生长的限制性商业惯例的兴趣。作为对照，收获细胞生长无4SU此外。另外3卷Trizol试剂（Sigma公司）洗细胞颗粒制造商的指示隔离的总RNA。进一步净化，用Qiagen公司的RNeasy根据制造商的协议的总RNA。为了防止在RNA提取和分析4SU氧化，添加0.1毫米二硫苏糖醇（DTT）洗缓冲区和随后的酶步骤。精华和去磷酸化的高效液相色谱分析的单核苷的总RNA作为描述的前 20名。简单地说，在30μL体积，孵育16小时40微克纯化的总RNA在37 °与0.4 ü细菌碱性磷酸酶（顿生化）和0.09 ü蛇毒磷酸二酯酶（顿生化）。作为参考标准，使用一个4SU标记合成的RNA（标准，我们使用CGUACGCGGAAUACUUCGA（4SU）美国），并受到完整的酶消化。一个Supelco公司发现C18（保税相硅胶5微米的颗粒，250 × 4.6毫米）反相柱（贝尔丰特PA，USA），高效液相色谱法分离核糖产生的混合物。高效液相色谱缓冲区0.1 M的TEAA在3％乙腈（A）和90％乙腈（二）在水中。 15分钟，0到10％B级为20分钟，30分钟10至100％B级，使用等度梯度：0％B级。应用5分钟100％B级洗应用之间运行清洁HPLC柱。 代表性的成果<img src="/files/ftp_upload/2034/2034fig1.jpg" alt="图1" /> 图1（右面板）显示了一个细胞系中表达的HA - 4 - SU和6 - SG标签IGF2BP1旗/执行PAR剪辑的代表性的结果。需要注意的是6 - SG的IGF2BP1的交联效率低于4苏交联效率。交联效率低，会导致在一个较高的背景丰富的细胞的RNA片段所得的序列，因此，你应该考虑扩大实验时，使用效率较低的光反应核苷。 图1的左侧面板中显示了使用不同的光反应尿嘧啶核苷类似物，可能是潜在的PAR剪辑使用，比传统的紫外线254纳米交联的比较。 正确长度的放射性波段的强度洞剪辑实验是否曾通过一个小RNA测序协议（小cDNA文库制备的说明一步一步您已经分离出了足够的RNA进行的给你一个好主意19），可以发现的RNA测序。当使用4 -苏和G A转换时使用6 - SG的特征突变测序读取，T到彗星转换，频率是成功交联的序列指示。根据我们的经验与4 -苏标记uncrosslinked RNA的一个背景的突变率约20％。这个比率是增加至约。后交联的50-80％。

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+composition+analysis+of+nucleosides+using+HPLC.">Base composition analysis of nucleosides using HPLC.</a> Current Protocols in Nucleic Acid Chemistry. Chapter 10 (Unit 10.6), (2001).</li></ol>

TT是一个Alnylam制药的创始人和科学顾问和顾问轩辕治疗。

缓冲液和试剂 HEK293细胞生长培养基<ul><li> DMEM培养液</li><li> 10％FBS </li><li> 2毫米L -谷氨酰胺</li><li> 100 U / ml青霉素</li><li> 100 U / ml链霉素</li><li> 100微克/毫升潮霉素</li><li> 15微克/毫升杀稻瘟</li></ul> 4 thiouridine股票的解决方案（1米） <ul><li> 260.27毫克4 thiouridine </li><li> 1毫升DMSO </li></ul> Doxycyclin股票（10毫克/毫升） <ul><li> 10毫克doxycyclin </li><li> 1毫升DMSO </li></ul> 1X NP40裂解液准备好没有DTT和蛋白酶抑制剂股票的5倍缓冲。实验前直接加入DTT和蛋白酶抑制剂。 <ul><li> 50毫米的HEPES，pH值7.5 </li><li> 150 mM的氯化钾</li><li> 2毫米EDTA </li><li> 1 mM的氟化钠</li><li> 0.5％（V / V）NP40 </li><li> 0.5 mM的数码地面电视</li><li>完整的EDTA无蛋白酶抑制剂的鸡尾酒（罗氏） </li></ul>柠檬酸 - 磷酸盐缓冲液<ul><li> 4.7克/升柠檬酸</li><li> 9.2克/升的NA 2 HPO 4 </li><li> pH值5.0 </li></ul> IP缓冲液洗<ul><li> 50毫米的HEPES - KOH，pH值7.5 </li><li> 300 mM的氯化钾</li><li> 0.05％（V / V）NP40 </li><li> 0.5毫米DTT（实验前直接添加） </li><li>完成EDTA无蛋白酶抑制剂的鸡尾酒（罗氏）（实验之前，直接添加） </li></ul>高盐缓冲液洗<ul><li> 50毫米的HEPES - KOH，pH值7.5 </li><li> 500 mM的氯化钾</li><li> 0.05％（V / V）NP40 </li><li> 0.5毫米DTT（实验前直接添加） </li><li>完成EDTA无蛋白酶抑制剂的鸡尾酒（罗氏）（实验前直接添加） </li></ul>去磷酸化缓冲<ul><li> 50毫米的Tris - HCl，pH值7.9 </li><li> 100毫米氯化钠</li><li> 10毫米氯化镁2 </li><li> 1 mM的数码地面电视</li></ul>磷酸酶清洗液<ul><li> 50毫米的Tris - HCl，pH值7.5 </li><li> 20 mM的EGTA </li><li> 0.5％（V / V）NP40 </li></ul>多聚核苷酸激酶（PNK）没有数码地面电视的缓冲区<ul><li> 50毫米的Tris - HCl，pH值7.5 </li><li> 50 mM氯化钠</li><li> 10毫米氯化镁2 </li></ul> PNK缓冲<ul><li> 50毫米的Tris - HCl，pH值7.5 </li><li> 50 mM氯化钠</li><li> 10毫米氯化镁2 </li><li> 5 mM的数码地面电视</li></ul> SDS - PAGE上样缓冲液<ul><li> 10％甘油（V / V） </li><li> 50毫米的Tris - HCl，pH值6.8 </li><li> 2毫米EDTA </li><li> 2％SDS（W / V） </li><li> 100 mM的数码地面电视</li><li> 0.1％溴酚蓝</li></ul> 2X蛋白酶K缓冲<ul><li> 100毫米的Tris - HCl，pH值7.5 </li><li> 150 mM氯化钠</li><li> 12.5毫米EDTA </li><li> 2％（W ​​/ V）SDS </li></ul>

par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins

通过数百个RNA结合蛋白（限制性商业惯例）和microRNA往往是在一个细胞类型依赖性表达的核蛋白复合物（miRNPs）。的相互作用，RNA转录，转录后基因调控要了解这些RNA结合因素的相互作用影响个人成绩单，在体内蛋白质的RNA的相互作用是必要的1的高分辨率地图的监管。 遗传，生化和计算方法相结合的典型应用，识别RNA - RBP的或RNA - RNP的相互作用。芯片分析immunopurified限制性商业惯例的RNA（RIP - Chip）的定义在转录水平的目标，但其应用受到限制的表征动力学稳定的相互作用和3,4只在极少数情况下可以识别的限制性商业惯例的识别元素（ RRE ）内的长期目标RNA。更直接的RBP的目标网站的信息是通过在体内的紫外光交联的交联的RNA片段和基因测序（CLIP）10隔离与免疫 7-9 5,6结合。剪辑是用来识别一个限制性商业惯例的11-17的目标。然而，剪辑是有限的紫外线254纳米RNA -蛋白质交联效率低，交联的位置是不容易识别的内交联的序列片段，使其难以从背景中分离非交联紫外光交联的靶RNA片段RNA片段也存在于样品中。 我们制定了一个强大的细胞为基础的交联的方法，以确定在高分辨率和转录的全细胞限制性商业惯例和miRNPs的结合位点，我们长期的PAR -剪辑（Photoactivatable -核苷增强的交联和免疫）（见大纲图1a的方法）。该方法通过活细胞的光反应的核苷类似物，如4 - thiouridine（4 - SU）和6 thioguanosine（6 - SG），纳入到新生RNA转录依赖。 365纳米的紫外线光照射诱导细胞光反应核苷标记细胞的RNA相互作用的限制性商业惯例的有效交联。其次是隔离交联和coimmunoprecipitated RNA免疫沉淀的限制性商业惯例的利益。分离出的RNA转换成cDNA文库和使用深测序公司Solexa技术。 PAR剪辑编写的cDNA文库的一个特点是，可以由居住在序列的cDNA突变确定的交联的精确位置。当使用4 -苏交联序列胸苷胞苷过渡，而使用6 - SG的鸟苷腺苷基因突变的结果。交联序列的突变，使人们有可能分开他们从丰富的细胞分子RNA的序列的背景。 据报道方法应用到一个多样化的RNA结合蛋白的数量在哈夫纳等 18

Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA转录受到广泛的是由众多的反式作用的RNA结合蛋白（限制性商业惯例）介导的转录后调控。在这里，我们提出了一个普及的方法，准确地确定和一个转录大规模RNA结合位点的限制性商业惯例。

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

par-clip-method-to-identify-transcriptome-wide-binding-sites-rna

Nanyang Technological University

Research

JoVE Journal

Biology

53.3K 次观看.  Rockefeller University. RNA转录受到广泛的是由众多的反式作用的RNA结合蛋白（限制性商业惯例）介导的转录后调控。在这里，我们提出了一个普及的方法，准确地确定和一个转录大规模RNA结合位点的限制性商业惯例。

视频: PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

收集可变浓度等温滴定量热数据集，以确定约束机制

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

钙调蛋白结合蛋白的下拉

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

摘要

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此视频中的章节

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

分析甲基化和其他 S腺苷_{L同型半胱氨酸结合蛋白}

收集可变浓度等温滴定量热数据集，以确定约束机制

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

钙调蛋白结合蛋白的下拉

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

核糖体束缚新生多肽的分离

蛋白质纯化方法测定结合亲和力微尺度热泳

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

摘要

探索更多视频

此视频中的章节

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

分析甲基化和其他 S腺苷 L同型半胱氨酸结合蛋白

收集可变浓度等温滴定量热数据集，以确定约束机制

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

钙调蛋白结合蛋白的下拉

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

核糖体束缚新生多肽的分离

蛋白质纯化方法测定结合亲和力微尺度热泳

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析

分析甲基化和其他 S腺苷_{L同型半胱氨酸结合蛋白}