Name: in vitro methylation assay to study protein arginine methylation
Uploaded: 2014-10-05T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about In vitro Methylation Assay to Study Protein Arginine Methylation at JoVE.com

这项研究是由美国退伍军人事务的美国国防部优异奖的支持，以及美国国立卫生研究院授予R01CA138528到RW。

1，制备表达构建<ol><li>启动协议之前，克隆PRMTs成具有N-末端表位标签（Myc基因或HA），并在帧与谷胱甘肽-S-转移酶（GST）的底物从细菌细胞中高水平的蛋白表达和纯化的哺乳动物表达载体裂解物，使用标准的分子生物学技术。 </li></ol> 2，净化PRMTs的<ol><li>使用100毫米培养皿中，以维持人体支气管上皮细胞（Beas2B细）。通过细胞每3天，不要让他们成长为汇合。 </li><li>于转染前一天，种子在100毫米培养皿7.5×10 5个细胞于10ml培养基中。漩涡均匀地分散细胞，并在5％增湿的CO 2培养箱中孵育在37℃。 </li><li>第二天，吸出介质并加入5 ml培养基不含血清和抗生素。漩涡，吸的媒介。重复此步骤1更多的时间，最后加入5毫升血清/无抗生素的培养基中，然后继续转。 </li><li>稀释6微克质粒DNA进入750μl的无血清培养基在无菌1.5毫升塑料离心管中。 </li><li>在另外150毫升的塑料离心管中，稀释30微升转染试剂的成750μl的无血清培养基。 </li><li>传送稀释转染试剂溶液（步骤2.5），以含有稀释的DNA（步骤2.4）的管中。移液器向上和向下几次轻轻混匀。 </li><li>在室温下孵育转染混合物15分钟。 </li><li> 15分钟后，吸取了转用文火到细胞，漩涡混合均匀，并以5％的潮湿二氧化碳培养箱在37℃。 </li><li> 3小时后，吸出含有细胞的转染混合物的介质。添加补充有血清和抗生素的新鲜细胞培养基。 </li><li>在5％的潮湿的CO 2在37℃下培养箱中培养48小时。 </li><li> 48小时后，裂解细胞在1ml裂解缓冲液（1×磷酸缓冲盐溶液，1％诺乃洗涤剂P-40，0.5％脱氧胆酸钠，0.1％十二烷基硫酸钠，和1微克/毫升phenlymethyl磺酰氟）。收集裂解物转移至塑料离心管中的细胞挺杆和孵化的溶胞产物在冰上30分钟。 </li><li>离心裂解液在15000 XG用于在台式离心10分钟，以清除细胞碎片的裂解液。 [*强烈建议在进行到酶的甲基化检测的纯化之前进行确认的HA-PRMT1通过用抗-HA抗体的裂解物中，等分试样的免疫印迹的表达。] </li><li>为PRMTs的纯化，孵育细胞裂解物的3毫克与30微升50:50悬浮抗HA亲和基质的磷酸缓冲盐水（PBS）中，并旋转过夜，在4℃在试管旋转器。 </li><li>第二天，旋管在14000 XG在台式离心机2分钟收集珠。弃上清，洗涤珠子3倍用1ml RIPA缓冲液〔20毫摩尔Tris（pH值8.0），150 mM氯化钠，5mM的EDTA和1％的Triton-X-100]在每次洗涤后离心以14,000 xg离心2分钟。在第三次洗涤后，用100μl的甲基化缓冲一次洗珠（50毫摩尔Tris pH值8.5，20mM的氯化钾，10mM的MgCl 2的，1mM的β-巯基乙醇，100mM的蔗糖）。离心后，除去上清液。含有固定化的PRMTs珠子是准备在甲基化检测中使用。 [*强烈建议在甲基化检测，立即用固定PRMTs。] </li></ol> 3，净化基质<ol><li>接种GST-G3BP1的单菌落转化BL21到5毫升的Luria-Bertani（LB）培养基中含有100微克/毫升氨苄青霉素。过夜生长的培养物在37℃下以250rpm摇动。 </li><li>第二天，过夜培养转移到50毫升的新鲜LB培养基中，并续inue增长到外径600的0.5-0.7（约需2小时）。 </li><li>当OD 600达到0.5-0.7，在37℃下加入异丙基β-D-半乳糖苷（IPTG）至1毫米的终浓度，继续培养更多的4小时用在250转摇。 </li><li>收获细胞，离心，在1500×g离心10分钟，并处理该沉淀为GST纯化或储存于-80℃。 </li><li>重悬细菌沉淀在5ml裂解缓冲液（1×PBS中与蛋白酶抑制剂，溶菌酶的100微克/毫升和0.1％的Triton-X-100）和孵育的裂解液在37℃下进行30分钟。此时的裂解物中会出现混浊和粘稠由于细菌的基因组DNA的释放。 </li><li>剪切通过超声处理的基因组DNA（10个脉冲，分别使用30％的幅度1秒）。 </li><li>清除裂解物离心以15,000 xg离心10分钟，将上层清液转移到15毫升管。如果上清液中是不明确的重复centrifugation过程一次。 </li><li>同时，用冰冷的PBS洗两次的GST-琼脂糖珠，并以50:50悬浮GST-琼脂糖凝胶在PBS中。加入200μl的GST-琼脂糖悬浮液中，以被清除的细菌细胞裂解液并旋转，在4℃下反应1小时。 </li><li> 1小时后，离心管中，在500×g离心3分钟以收集珠子。洗珠3倍用冰冷的PBS中。 </li><li>最后一次洗涤后，重悬珠子在100μl洗脱缓冲液（50mM的Tris pH值8.0，150mM的氯化钠，30mM的还原型谷胱甘肽）和旋转，在4℃下进行10分钟。 </li><li>离心管中以15,000 xg离心2分钟，并含有纯化蛋白的上清液转移到另一塑料离心管中。进行SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）分析5微升的纯化蛋白沿着与已知量的牛血清白蛋白（BSA）来估计纯化的蛋白质的浓度。 </li></ol> 4，甲基化分析注:请在指定的放射性同位素工作，并符合上进行实验，放射性同位素的机构的协议的一个单独的区域下面的步骤。 <ol><li>加入2微克纯化的GST-基质与含有1x甲基化缓冲液（50mM的Tris pH值8.5，20mM的氯化钾，10mM的MgCl 2的，1mM的β-巯基乙醇，100mM的蔗糖），1微居里的反应混合液的S-腺苷- L-[甲基- 3 H] -甲硫氨酸，和水加至10微升的固定PRMTs（步骤2.13），使终体积和孵育在30℃下反应1小时。 </li><li> 1小时后，停止通过加入10μl的2×SDS样品缓冲液中反应。样品的变性，在95℃下进行10分钟后，分离出的样品在SDS-PAGE凝胶上。 </li><li>以后，通过利用半干电泳转移从SDS凝胶转移分离的蛋白质到硝酸纤维素膜上。 </li><li>转让完成后，盖上膜utofluor（放射自显影图像增强器），并轻轻摇动2小时，在室温下进行。 2小时后，倒出autofluor并保留以备后用。 [*标签管的放射性物质] </li><li>很快风干膜滤纸上，密封在塑料袋中，露出膜的X光片在-20℃。 5-30天之间有足够的曝光时间范围。 </li></ol> 5，确认PRMT表达和平等基质加载中<ol><li>获得足够的曝光后，将膜转移至塑料盒含有封闭缓冲液[Tris缓冲盐水和吐温20（TBST +3％脱脂乳）并轻轻摇动1小时。 </li><li>弃去封闭缓冲液作为放射性废物。孵化用抗HA抗体的膜:在TBST（1千稀释）过夜，在4 ℃，用温和的摇摆。 </li><li>洗用TBST 3倍的膜孵育与HRP标记的抗小鼠二抗的膜（1:5000稀释）在TBST中于室温下1小时，轻轻摇动。 </li><li> 1小时后，用TBST洗涤3次洗膜，并进行化学发光检测的蛋白质与HRP底物。 </li><li>检测HA标记的PRMTs后，孵育Ponseau-S染色溶液中的膜5分钟，在室温下进行。以水快速洗膜3次，以检测GST标记的底物。扫描图像。 </li></ol>

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	<li>Paik, W. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+methylase+I.+Purification+and+properties+of+the+enzyme.">Protein methylase I. Purification and properties of the enzyme.</a> J Biol Chem. 243, 2108-2114 (1968).</li><li>Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+immediate-early+TIS21+protein+and+the+leukemia-associated+BTG1+protein+interact+with+a+protein-arginine+N-methyltransferase.">The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase.</a> J Biol Chem. 271, 15034-15044 (1996).</li><li>Boffa, L. C., Karn, J., Vidali, G., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+NG,+NG,-dimethylarginine+in+nuclear+protein+fractions.">Distribution of NG, NG,-dimethylarginine in nuclear protein fractions.</a> Biochem Biophys Res Commun. 74, 969-976 (1977).</li><li>Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+at+a+glance.">Arginine methylation at a glance.</a> J Cell Sci. 120, 4243-4246 (2007).</li><li>Lee, Y. H., Stallcup, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+protein+arginine+methylation+of+nonhistone+proteins+in+transcriptional+regulation.">Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.</a> Mol Endocrinol. 23, 425-433 (2009).</li><li>Bedford, M. T., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Mol Cell. 33, 1-13 (2009).</li><li>Bikkavilli, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled3+is+a+novel+arginine+methyl+transferase+substrate.">Dishevelled3 is a novel arginine methyl transferase substrate.</a> Scientific reports. 2, 805 (2012).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+of+G3BP1+in+response+to+Wnt3a+regulates+{beta}-catenin+mRNA.">Arginine methylation of G3BP1 in response to Wnt3a regulates {beta}-catenin mRNA.</a> J Cell Sci. 124, 2310-2320 (2011).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a-stimulated+LRP6+phosphorylation+is+dependent+upon+arginine+methylation+of+G3BP2.">Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.</a> J Cell Sci. , (2012).</li><li>Tini, M., Naeem, H., Torchia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+arginine+methylation+in+transcription.">Biochemical analysis of arginine methylation in transcription.</a> Methods Mol Biol. 523, 235-247 (2009).</li><li>Lee, J., Cheng, D., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+protein+methylation.">Techniques in protein methylation.</a> Methods Mol Biol. 284, 195-208 (2004).</li><li>Cheng, D., Vemulapalli, V., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+applied+to+the+study+of+protein+arginine+methylation.">Methods applied to the study of protein arginine methylation.</a> Methods Enzymol. 512, 71-92 (2012).</li><li>Zurita-Lopez, C. I., Sandberg, T., Kelly, R., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+protein+arginine+methyltransferase+7+(PRMT7)+is+a+type+III+enzyme+forming+omega-NG-monomethylated+arginine+residues.">Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues.</a> J Biol Chem. 287, 7859-7870 (2012).</li></ol>

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本文所描述的协议通常用于建立所标识的PRMT底物的甲基化状态。这一功能强大且一致的分析也将提供PRMTs其底物的特异性确切的证据。关键部件的该测定法的成功是:PRMTs在哺乳动物细胞中的表达1，2，活性纯化的PRMTs的，3的表达和衬底的纯化和蛋白质的4完全西方转印到硝酸纤维素膜上。 重组PRMTs表达，并从细菌中纯化的，也可以在体外甲基化反应10,11,13使用。?...

蛋白甲基化是在1968年1首先描述。直到PRMT1在1996年的第一个克隆，研究人员开始欣赏这种翻译后修饰2的重要性。有趣的是，约2％精氨酸残基在核提取物中的蛋白质被甲基3，说明本变形例的丰度。精氨酸是一种带正电荷的氨基酸与碱性侧链和氮/ s的精氨酸的侧链中可以在翻译后通过加入甲基，称为精氨酸甲基4-6的过程的修改。精氨酸甲基化的一类酶即催化，蛋白质精氨酸甲基转移酶（PRMTs）。精氨酸既可以单甲基化或二甲基化，而 ​​后者可以是对称或不对称的视PRMTs催化过程4-6的类型。 这显示精氨酸的蛋白质E-甘氨酸丰富的图案是PRMT介导的催化潜在目标。基板的PRMT介导的甲基化已显示调节蛋白质-蛋白质相互作用，蛋白质-核酸相互作用，蛋白质功能，基因表达，和/或细胞信号，所有这些都对正常细胞稳态7-9关键。为了理解的蛋白质精氨酸甲基化的生物作用，精确，高效和可重复的试验，需要建立所标识的PRMT底物的甲基化状态。 在体外甲基化检测，评估其纯化PRMTs的能力，以促进它们的底物的甲基化，是研究精氨酸甲基化10-12广为接受检测。此测定法的总体成功在很大程度上依赖于纯化的PRMTs的活性。 PRMTs可以表示和从细菌或哺乳动物细胞10,11纯化。此体外甲基化检测，为detailed的协议部分，是基于最初由蒂尼和他的同事10中描述的方法。在这个协议中，我们显示了详细涉及PRMT1在哺乳动物细胞中的表达和纯化的步骤。纯化PRMT1的催化甲基上的Ras-GTP酶激活蛋白结合蛋白1（G3BP1），一种已知的PRMT底物8的能力中的S-腺苷-L-甲基- 3 H] -甲硫氨酸的存在下作为后来评价甲基供体。使用这种方法，我们能够可靠地限定PRMTs的能力甲醇新颖的或已知的底物，这是在蛋白质精氨酸甲基化的研究的主要步骤。

<table><tbody><tr><td>S-adenosyl-L-[methyl-&lt;sup&gt;3&lt;/sup&gt;H] methionine</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Ecoscint ultra&amp;nbsp;</td><td>National Diagnostics</td><td>LS-270</td><td></td></tr><tr><td>Bottle top dispenser</td><td>National Diagnostics</td><td>LS-900</td><td></td></tr><tr><td>AutoFluor</td><td>National Diagnostics</td><td>LS-315</td><td></td></tr><tr><td>6 ml Scintillation Vials</td><td>National Diagnostics</td><td>SKU:SVC-06</td><td></td></tr><tr><td>GST-sepharose</td><td>GE Life Sciences</td><td></td><td></td></tr><tr><td>L-Glutathione Reduced</td><td>Sigma</td><td>G4251</td><td></td></tr><tr><td>Lipofectamine</td><td>Invitrogen</td><td></td><td>Transfection reagent</td></tr><tr><td>Beas2B</td><td>ATCC</td><td></td><td></td></tr><tr><td>Optimem</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>NP-40</td><td>US-Biologicals</td><td></td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td></td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td></td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td></td><td></td></tr><tr><td>anti-HA affinity matrix</td><td>Roche</td><td></td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td></td><td></td></tr><tr><td>Nacl</td><td>Sigma</td><td></td><td></td></tr><tr><td>Bio-rad gel doc</td><td>Bio Rad</td><td></td><td></td></tr><tr><td>anti-HA antibody</td><td>roche</td><td></td><td></td></tr><tr><td>Ponseau S</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

in vitro methylation assay to study protein arginine methylation

蛋白质精氨酸甲基化是在细胞核中最丰富的翻译后修饰之一。蛋白质精氨酸甲基可以被识别和/或通过蛋白质组的方法来确定，和/或以甲基精氨酸特异性抗体进行免疫印迹。然而，这些技术有时可能会产生误导，并经常提供假阳性结果。最重要的是，这些技术不能提供支持的PRMT底物特异性的直接证据。 体外甲基化分析，在另一方面，是有用的生化分析，这是敏感的，并且始终如一地揭示，如果所识别的蛋白确实PRMT底物。一个典型的体外甲基化检测包括纯化的活性PRMTs，纯化的底物和放射性同位素标记的甲基供体（S-腺苷-L-甲基- 3 H] -甲硫氨酸）。在这里，我们描述了一个一步一步的协议隔离催化活性PRMT1一个广泛表达PRMT家族成员。在我纯化PRMT1的基苯乙基转移酶活性上的Ras-GTP酶激活蛋白结合蛋白1（G3BP1），一种已知的PRMT底物后进行测试，在S-腺苷-L-甲基- 3 H] -甲硫氨酸作为甲基供体的存在下进行。这个协议可以采用不仅用于建立的新的生理PRMT1基板的甲基化状态，而且对于了解蛋白质精氨酸甲基化的基本机制。

<html> HA-PRMT1的能力，以甲基化的GST-G3BP1由体外甲基化测定法测定。 HA标记PRMT1从Beas2B细细胞溶胞产物纯化被用在含有GST-G3BP1甲基化反应，和1微居里的S-腺苷-L-甲基- 3 H] -蛋氨酸，作为甲基供体。 PRMT1能有效地催化GST-G3BP1（ 图1，上图）的甲基化。使用上作为阴性对照组（ 图1，上图，泳道1）转染空载体Beas2B细细胞裂解液进行拉起伏甲基化反应。免疫?...

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In vitro Methylation Assay to Study Protein Arginine Methylation

蛋白质精氨酸甲基化，由一类酶即催化。蛋白质精氨酸甲基转移酶（PRMTs），是酶促加入甲基组（S），以蛋白质内精氨酸的过程。 在体外甲基化测定法是最可靠的工具，以评估已知的或新型的PRMT底物的甲基化状态。

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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Research

JoVE Journal

Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.2K 次观看.  University of Illinois at Chicago. 蛋白质精氨酸甲基化，由一类酶即催化。蛋白质精氨酸甲基转移酶（PRMTs），是酶促加入甲基组（S），以蛋白质内精氨酸的过程。 在体外甲基化测定法是最可靠的工具，以评估已知的或新型的PRMT底物的甲基化状态。 

视频: In vitro Methylation Assay to Study Protein Arginine Methylation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA甲基转移酶活性分析的无抗体测定

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

遗传学

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

用核磁共振波谱探索精氨酸甲基组

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

癌症研究

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

生物化学

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

研究蛋白质精氨酸甲基转移酶1-9细胞活性的方法

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...