我们感谢Karyn Esser，Nobuya小池和Noheon Park提供有用的建议。这项工作部分由NIH / NIGMS（R01GM114424）向S.-HY和Robert A.Welch基金会（AU-1731）和NIH / NIA（R01AG045828）向ZC提供支持

动物护理在机构动物护理和使用委员会（IACUC）指南下进行，并且程序是根据得克萨斯州休斯敦大学健康科学中心批准的动物方案进行的。 从交联骨骼肌中分离核<ol><li>称重和切碎后肢骨骼肌，从大约20周龄的C57BL / 6雄性小鼠分离，在冰冷的磷酸盐缓冲盐水（PBS）中，如先前详细描述的22所述 。 注意:我们通常从一只小鼠获得1.0 - 1.5 g骨骼肌。 <ol><li>将切碎的骨骼肌组织放入含有10mL PBS的50mL锥形管中。 </li></ol></li><li>在4℃下以300xg离心5分钟。 </li><li>通过抽吸小心清除PBS上清液。 </li><li>通过使用具有1,2,3和4mL水的参考管估计每个50mL管中的颗粒体积。 </li><li>添加7卷冰冷的1％甲醛在PBS中，并使用探针组织匀浆器在冰上匀化样品（ 参见表材料 ）。 注意:可选:均质程序可在冷藏室进行。 </li><li>通过在室温下孵育10分钟将样品交联。 </li><li>通过加入1M甘氨酸使终浓度为0.125M的交联反应骤冷，并在室温下孵育5分钟。 </li><li>在4℃下将样品以3,000xg离心5分钟。通过抽吸去除上清液。 </li><li>用含有新鲜蛋白酶抑制剂（0.2mM苯甲基磺酰氟（PMSF））的10mL冰冷碱性缓冲液（pH7.3的10mM HEPES-KOH，10mM EDTA，10mM KCl，5mM MgCl 2，0.5mM DTT） ，和10μg/ mL亮抑酶肽）。在4℃下以3,000g离心5分钟，并通过抽吸小心地去除上清液。 </li><li>将沉淀重悬于6mL裂解缓冲液（10mM HEPES-KOH，含有新鲜蛋白酶抑制剂（0.2mM PMSF和10μg/ mL亮抑酶肽）的H 7.3，10mM EDTA，10mM KCl，5mM MgCl 2，0.5mM DTT，1％Triton X-100）。 </li><li>将样品转移到预先制备的15 mL Dounce匀浆器中，并在冰上孵育10分钟。在冰上均匀化每个样品，使用松散的杵进行15次缓慢的冲程，然后用紧密的杵将15次冲击以释放细胞核。 </li><li>通过细胞过滤器（孔径:100μm）过滤匀浆（6 mL），并用4 mL裂解缓冲液冲洗。在4℃下将样品以1,000xg离心10分钟。重悬于5 mL冰冷的基础缓冲液中，用紧密的杵将其重复10次以释放细胞核。 <ol><li>用分光光度计（OD260 / OD280）取出20μL的DNA浓度测定等分试样，必要时用台盼蓝进行显微镜观察（见下文）（ 图 1A ）。 </li></ol></li><li>过滤悬浮液细胞过滤器（孔径:70μm），如上所述用2mL碱缓冲液再次冲洗管并过滤。 </li><li>重复过滤步骤1.13，细孔过滤器逐渐减小孔径（40μm，30μm，20μm和10μm）。 注意:在步骤1.12-1.14中总共使用6孔径的过滤器。 </li><li>在4℃下以1,000g离心10分钟。去除上清液，并将沉淀物重悬于500μL碱性缓冲液中。 </li><li>测量如上所述的DNA浓度。 注意:从一只动物的两只后肢组合获得约200μg的核酸DNA。任选地，用台盼蓝染色（库存浓度0.4％，1:5稀释）显微镜观察保存20μL;核会染蓝色（ 图1B ）。 </li><li>在4℃下以1,000g离心10分钟，弃上清。将沉淀物储存在-80°C。 </li></ol>超声处理 &lt;OL&gt; <li>在500μLSDS裂解缓冲液中解冻样品，并轻轻移液重悬。 </li><li>将核DNA悬浮液转移到冰上的玻璃小瓶中。 </li><li>从碟形传感器运行超音波超声波声能。有关设备详细信息，请参见材料表 。使用以下设置:每个突发周期:200;强度:5;占空比:20;温度4-6℃; 30秒开/关。 注:评估超声功效的实例如图2所示。 </li><li>将超声处理的染色质转移到1.5 mL管中。以12,000 xg离心15分钟，并将上清液转移到新管中。将50μL超声处理的样品（〜7 - 10μg/肌肉样品）转移到新的1.5 mL管中，以在65°C进行反向交联过夜。将剩余的样品在-80℃下冷冻。 </li></ol>超声和定量评估<ol><li>添加1.0μL的RNA酶A（500 U / mL RNA酶A:20,000 U / mL RNase T1）保存在50μL超声波样品中，37℃孵育30 min。 </li><li>加入8μL蛋白酶K（10mg / mL），并在55℃下孵育30分钟。 </li><li>通过DNA提取试剂盒收获DNA。 <ol><li>加入5卷结合缓冲液，应用于旋转柱，离心4,000 xg，10分钟。 </li><li>将通过应用到同一列并再次离心。 </li><li>用洗涤缓冲液洗涤两次，每次13,000 xg，1分钟。 </li><li>离心再次以13,000 xg，1分钟干燥柱。 </li><li>加入50μLH 2 O，离心13000 x g，1 min洗脱DNA。 </li><li>将通过应用到同一列并再次离心。 </li></ol></li><li>用分光光度计（OD260 / OD280）定量DNA量。在0.8％凝胶上运行样品以评估超声产品的大小和量（1μg）（ 图2 ）。 注意:平均染色质产量约为〜20％。 </li></ol> ChIP <ol><li>将以前保存的染色质在-80℃下解冻，并将染色质等分至〜100〜120μg/管。使用放射免疫沉淀测定（RIPA）缓冲液（20mM Tris-HCl（pH 7.4），150mM NaCl，1mM EDTA，1mM NaF，1mM Na 3 VO 4，1mM PMSF，1mM蛋白酶抑制剂混合物（PIC），0.1％脱氧胆酸盐w / v），1％Triton X-100）。 <ol><li>如果每组中有多个样品，则将等量的染色质DNA组合至最终量为100 - 120μg/样品。节省10％的输入。 </li></ol></li><li>加入BSA-pre-blocked（〜2 h，4℃）蛋白A / G琼脂糖（非鸡抗体）或IgY珠（鸡抗体）的40μL（RIPA缓冲液中的50％v / v浆液），并孵育在4℃下旋转3小时。 </li><li>以1,000 xg，10分钟离心，小心地将上清液转移到新管中。 </li><li>每1μg抗体加1μg抗体25-100μg染色质DNA。 </li><li>以约15rpm在4℃下轻轻旋转过夜。 </li><li>加入10μLBSA-预封闭（〜2 h，4°C）蛋白A / G琼脂糖（非鸡抗体）或IgY珠（鸡抗体）混合，并在冷藏室中旋转2 h。 </li><li>洗珠如下。用1mL RIPA缓冲液洗涤3分钟两次，用1mL高盐缓冲液（20mM Tris-HCl（pH7.4），500mM NaCl，2mM EDTA，1mM PMSF，1％Triton X-100）洗涤3分钟两次，用1mL LiCl缓冲液（20mM Tris-HCl（pH7.4），250mM LiCl，2mM EDTA，1mM PMSF，1％脱氧胆酸钠，0.5％NP40）洗涤3分钟两次。 <ol><li>用1mL Tris-EDTA（TE）缓冲液（10mM Tris-HCl（pH7.4），1mM EDTA）洗涤3分钟一次。然后用洗脱缓冲液（20mM Tris-HCl，5mM EDTA和0.5％SDS）洗脱DNA。 </li></ol></li><li>以1,000 g离心1分钟，小心取出上清液。 </li><li>用50μL洗脱缓冲液重悬珠。 </li><li>孵化10min，65℃。 </li><li>以12,000克离心5分钟。 </li><li>将上清液转移到新管中，再加入50μL，再次以12,000 g离心5分钟。最终洗脱体积为100μL。 </li><li>反向交联和DNA洗脱。 <ol><li>将洗脱的染色质在65℃孵育过夜。 </li><li>加入1.0μLRNA酶A，37℃孵育30 min。 </li><li>加入8μL蛋白酶K（10mg / mL），并在55℃下孵育30分钟。 </li><li>通过使用PCR清洗试剂盒，根据制造商的方案，以50μL的洗脱体积收获DNA。 </li></ol></li><li>通过实时qPCR分析配置文件，如前所述25,26 。 注意:引物序列如图3所示 。数据以平均值±SEM表示（ 图3 ）。 </li></ol>

<ol>
	<li>Bouzakri, 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=siRNA-based+gene+silencing+reveals+specialized+roles+of+IRS-1/Akt2+and+IRS-2/Akt1+in+glucose+and+lipid+metabolism+in+human+skeletal+muscle.">siRNA-based gene silencing reveals specialized roles of IRS-1/Akt2 and IRS-2/Akt1 in glucose and lipid metabolism in human skeletal muscle.</a> Cell Metab. 4 (1), 89-96 (2006).</li><li>Boucher, J., Kleinridders, A., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+receptor+signaling+in+normal+and+insulin-resistant+states.">Insulin receptor signaling in normal and insulin-resistant states.</a> Cold Spring Harb Perspect Biol. 6 (1), (2014).</li><li>Green, C. B., Takahashi, J. S., Bass, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+meter+of+metabolism.">The meter of metabolism.</a> Cell. 134 (5), 728-742 (2008).</li><li>Liu, A. C., Lewis, W. G., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+circadian+signaling+networks+and+therapeutic+targets.">Mammalian circadian signaling networks and therapeutic targets.</a> Nat Chem Biol. 3 (10), 630-639 (2007).</li><li>Harfmann, B. D., Schroder, E. A., Esser, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms,+the+molecular+clock,+and+skeletal+muscle.">Circadian rhythms, the molecular clock, and skeletal muscle.</a> J Biol Rhythms. 30 (2), 84-94 (2015).</li><li>Nohara, K., Yoo, S. H., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+the+circadian+and+sleep+cycles+to+protect+against+metabolic+disease.">Manipulating the circadian and sleep cycles to protect against metabolic disease.</a> Front Endocrinol (Lausanne). 6, 35 (2015).</li><li>Harfmann, B. D., 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=Muscle-specific+loss+of+Bmal1+leads+to+disrupted+tissue+glucose+metabolism+and+systemic+glucose+homeostasis.">Muscle-specific loss of Bmal1 leads to disrupted tissue glucose metabolism and systemic glucose homeostasis.</a> Skelet Muscle. 6, 12 (2016).</li><li>Pedersen, B. K., Febbraio, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles,+exercise+and+obesity:+skeletal+muscle+as+a+secretory+organ.">Muscles, exercise and obesity: skeletal muscle as a secretory organ.</a> Nat Rev Endocrinol. 8 (8), 457-465 (2012).</li><li>Solomon, M. J., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-mediated+DNA-protein+crosslinking:+a+probe+for+in+vivo+chromatin+structures.">Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.</a> Proc Natl Acad Sci U S A. 82 (19), 6470-6474 (1985).</li><li>Jackson, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+histone+organization+in+the+nucleosome+using+formaldehyde+as+a+reversible+cross-linking+agent.">Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent.</a> Cell. 15 (3), 945-954 (1978).</li><li>Gilmour, D. S., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+protein-DNA+interactions+in+vivo:+distribution+of+RNA+polymerase+on+specific+bacterial+genes.">Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.</a> Proc Natl Acad Sci U S A. 81 (14), 4275-4279 (1984).</li><li>Takahashi, J. S., 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=ChIP-seq+and+RNA-seq+methods+to+study+circadian+control+of+transcription+in+mammals.">ChIP-seq and RNA-seq methods to study circadian control of transcription in mammals.</a> Methods Enzymol. 551, 285-321 (2015).</li><li>Kuo, M. H., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cross-linking+and+immunoprecipitation+for+studying+dynamic+Protein:DNA+associations+in+a+chromatin+environment.">In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment.</a> Methods. 19 (3), 425-433 (1999).</li><li>Chen, Z., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Core+promoter+elements+and+TAFs+contribute+to+the+diversity+of+transcriptional+activation+in+vertebrates.">Core promoter elements and TAFs contribute to the diversity of transcriptional activation in vertebrates.</a> Mol Cell Biol. 23 (20), 7350-7362 (2003).</li><li>Menet, J. S., Abruzzi, K. C., Desrochers, J., Rodriguez, J., Rosbash, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+PER+repression+mechanisms+in+the+Drosophila+circadian+clock:+from+on-DNA+to+off-DNA.">Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.</a> Genes Dev. 24 (4), 358-367 (2010).</li><li>Miki, T., Matsumoto, T., Zhao, Z., Lee, 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=p53+regulates+Period2+expression+and+the+circadian+clock.">p53 regulates Period2 expression and the circadian clock.</a> Nat Commun. 4, 2444 (2013).</li><li>Furey, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+beyond:+new+and+improved+methodologies+to+detect+and+characterize+protein-DNA+interactions.">ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.</a> Nat Rev Genet. 13 (12), 840-852 (2012).</li><li>Koike, N., 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=Transcriptional+architecture+and+chromatin+landscape+of+the+core+circadian+clock+in+mammals.">Transcriptional architecture and chromatin landscape of the core circadian clock in mammals.</a> Science. 338 (6105), 349-354 (2012).</li><li>Zhou, J., Yu, W., Hardin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPping+away+at+the+Drosophila+clock.">ChIPping away at the Drosophila clock.</a> Methods Enzymol. 551, 323-347 (2015).</li><li>Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+architecture+of+the+mammalian+circadian+clock.">Transcriptional architecture of the mammalian circadian clock.</a> Nat Rev Genet. , (2016).</li><li>Edelman, J. C., Edelman, P. M., Kniggee, K. M., Schwartz, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+nuclei.">Isolation of skeletal muscle nuclei.</a> J Cell Biol. 27 (2), 365-377 (1965).</li><li>Ohkawa, Y., Mallappa, C., Vallaster, C. S., Imbalzano, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+from+skeletal+muscle+satellite+cells+and+myofibers+for+use+in+chromatin+immunoprecipitation+assays.">Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays.</a> Methods Mol Biol. 798, 517-530 (2012).</li><li>Wilkie, G. S., Schirmer, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nuclei+and+preparation+of+nuclear+envelopes+from+skeletal+muscle.">Purification of nuclei and preparation of nuclear envelopes from skeletal muscle.</a> Methods Mol Biol. 463, 23-41 (2008).</li><li>Hahn, C. G., Covault, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+transcriptionally+active+nuclei+from+striated+muscle+using+Percoll+density+gradients.">Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients.</a> Anal Biochem. 190 (2), 193-197 (1990).</li><li>Jeong, 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=Dual+attenuation+of+proteasomal+and+autophagic+BMAL1+degradation+in+Clock+Delta19/++mice+contributes+to+improved+glucose+homeostasis.">Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Delta19/+ mice contributes to improved glucose homeostasis.</a> Scientific reports. 5, 12801 (2015).</li><li>Nohara, 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=Ammonia-lowering+activities+and+carbamoyl+phosphate+synthetase+1+(Cps1)+induction+mechanism+of+a+natural+flavonoid.">Ammonia-lowering activities and carbamoyl phosphate synthetase 1 (Cps1) induction mechanism of a natural flavonoid.</a> Nutrition &amp; metabolism. 12, 23 (2015).</li><li>Zhang, L., Rubins, N. E., Ahima, R. S., Greenbaum, L. E., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxa2+integrates+the+transcriptional+response+of+the+hepatocyte+to+fasting.">Foxa2 integrates the transcriptional response of the hepatocyte to fasting.</a> Cell Metab. 2 (2), 141-148 (2005).</li><li>Gade, P., Kalvakolanu, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay+as+a+tool+for+analyzing+transcription+factor+activity.">Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.</a> Methods Mol Biol. 809, 85-104 (2012).</li><li>Kondratov, R. V., 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=BMAL1-dependent+circadian+oscillation+of+nuclear+CLOCK:+posttranslational+events+induced+by+dimerization+of+transcriptional+activators+of+the+mammalian+clock+system.">BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system.</a> Genes Dev. 17 (15), 1921-1932 (2003).</li><li>Ripperger, J. A., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+CLOCK-BMAL1+binding+to+multiple+E-box+motifs+drives+circadian+Dbp+transcription+and+chromatin+transitions.">Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions.</a> Nat Genet. 38 (3), 369-374 (2006).</li><li>Laplante, M., 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=DEPTOR+cell-autonomously+promotes+adipogenesis,+and+its+expression+is+associated+with+obesity.">DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity.</a> Cell Metab. 16 (2), 202-212 (2012).</li><li>Blechman, J., Amir-Zilberstein, L., Gutnick, A., Ben-Dor, S., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolic+regulator+PGC-1alpha+directly+controls+the+expression+of+the+hypothalamic+neuropeptide+oxytocin.">The metabolic regulator PGC-1alpha directly controls the expression of the hypothalamic neuropeptide oxytocin.</a> J Neurosci. 31 (42), 14835-14840 (2011).</li></ol>

作者没有什么可以披露的。

在这里，我们描述了一种使用交联骨骼肌组织分离高质量细胞核的可靠方法。进行顺序过滤以有效地将细胞核与碎片分离，来自碟形换能器的超声波能量剪切染色质进行ChIP分析。结果显示BMAL1与靶启动子的昼夜节律时间特异性结合。 当交联发生时，ChIP可用于捕获基因组DNA上的实时蛋白质占用。为了利用这种潜力，我们旨在开发一种方法，允许在组织解剖时交叉骨骼肌，并简?...

骨骼肌在生理和行为中起重要作用。多核肌纤维由肌原纤维组成，其中肌动蛋白和肌球蛋白形成称为sarcomeres的功能单位以产生收缩力。骨骼肌也是身体中最大的代谢器官，占餐后葡萄糖摄入量的80％以上，调节胰岛素反应和代谢体内平衡1,2 。肌肉生理和代谢受到昼夜节律钟的限制，内在的生物计时器3,4,5,6。例如， Bmal1的骨骼肌特异性缺失，核心昼夜节律钟成分之一，导致骨骼肌中胰岛素抵抗和葡萄糖摄取减少，发现动物发生2型糖尿病7 。一世此外，骨骼肌也越来越被认为是内分泌器官8 ，分泌肌动蛋白以调节全身代谢和生理学。机械研究需要完全了解骨骼肌中的这些调节功能。 ChIP是描述DNA结合蛋白启动子募集的有力方法。最初开发了ChIP以鉴定染色质DNA 9,10上的核小体组织。据报道，使用甲醛，硫酸二甲酯或紫外线照射（UV） 11,12来交联蛋白质和染色质DNA的各种方法。甲醛交联是最常用的，保存染色质结构和DNA-蛋白相互作用9,13,14 。交联色谱通过超声处理切片并用针对感兴趣的特定DNA结合蛋白15,16的抗体进行免疫沉淀。近年来，已经开发了ChIP-sequencing（ChIP-seq），一种将ChIP与NGS相结合的方法，用于询问全基因组转录因子结合17 ，并且在某些情况下监测时间过程18,19,20的动态变化。例如，昼夜节律ChIP-seq研究已经显示出昼夜节律时钟组分和组蛋白标记物的基因组结合的高度编排的序列，其在整个〜24小时昼夜循环中驱动时间上精确的基因表达18 。 大多数可用的ChIP协议设计用于软组织（ 例如肝，脑等 ），并且很少已经出版用于硬组织，包括ke伸肌在富含纤维的骨骼肌中均匀化并分离高质量细胞核21是技术上的挑战，特别是对于需要交联的ChIP实验。在最近的肌肉ChIP研究22中 ，卫星细胞与肌纤维分离，并且通过涉及组织消化的延长的过程从两种细胞类型制备细胞核。在分离的核上进行甲醛交联之前，整个过程需要大约三个小时才能完成。虽然这种方法避免了交联肌纤维，这使得肌肉组织更有效地进行高效均匀化，并且能够产生高质量的细胞核，但是从组织收集到细胞核交联的显着的时间滞后引起了DNA变化的风险蛋白质相互作用。相比之下，大多数研究在实验治疗或组织收集后立即进行交联，以保存实时DNA-蛋白质n约束12 。在交联前核分离的第二个缺点是它排除了时间敏感的应用，例如通常以3-4小时间隔发生的昼夜样本采集。没有交联核，分离需要在解剖后立即进行，而交联样品可以在整个时间过程完成后一起处理，从而确保更大的实验一致性。 还报道了与未交联的骨骼肌分离核的其它方案。两项研究描述了使用梯度超速离心将细胞核与剩余的肌原纤维和细胞碎片分开23,24 。虽然蔗糖或胶体梯度超速离心对未交联的肌肉组织有效，但我们的实验表明，交联后，梯度超速离心不能将细胞从细胞分离出来ebris在梯度上。 因此，我们开发了一种使用交联的小鼠骨骼肌组织分离高质量核的方法。而不是梯度超速离心，我们设计了一种串联过滤方法来有效地将细胞核与碎片分离。经超声处理后，染色质样品成功应用于ChIP研究，显示BMAL1蛋白与靶启动子结合的昼夜节律模式。我们的方法可以广泛适用于肌肉组织的各种机械研究。

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			<td style="width:288px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Formaldehyde solution</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glycine</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">BP381-5</td>
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			<td height="20" style="height:20px;width:216px;">Trypan Blue solution (0.4%)</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RNase A</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">10109142001</td>
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			<td height="20" style="height:20px;width:216px;">Protease K</td>
			<td style="width:143px;">Sigma Aldrich</td>
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			<td height="57" style="height:57px;width:216px;">Chicken Anti-BMAL1 antibody</td>
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			<td style="width:288px;">Generated in chicken (Cocalico Biologicals) against antigen aa 318-579, and IgY was affinity purified using the same antigen.&#160;</td>
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			<td height="20" style="height:20px;width:216px;">Chicken IgY Precipitating Resin</td>
			<td style="width:143px;">GenScript</td>
			<td style="width:119px;">L00405</td>
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			<td height="20" style="height:20px;width:216px;">Equipment</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="38" style="height:38px;width:216px;">KINEMATICA&#160;Polytron PT2100 Benchtop Homogenizer</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-451-178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">15mL Dounce tissue grinder</td>
			<td style="width:143px;">Whearton</td>
			<td style="width:119px;">357544</td>
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			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 100&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-19</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 70&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 40&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 30 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50030-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 20 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50020-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 10 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50010-03</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Covaris S2 Focused-ultrasonicator</td>
			<td style="width:143px;">Covaris</td>
			<td style="width:119px;">Model S2</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Labquake&#160;</td>
			<td>Thermo Scientific</td>
			<td>C415110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD Microscope Camera</td>
			<td>&#160;Leica Microsystems</td>
			<td>DFC3000 G</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Reagent Kit</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DNA extraction kit</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:119px;">K0691</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Buffers</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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</table>

a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

染色质免疫沉淀（ChIP）是确定蛋白质结合染色质DNA的有效方法。然而，富含纤维的骨骼肌由于技术上难以隔离高质量的细胞核而且对肌原纤维的污染最小，因此一直是ChIP的挑战。以前的方案尝试在交联之前纯化细胞核，这导致在延长的细胞核制备过程中DNA-蛋白质相互作用发生改变的风险。在目前的方案中，我们首先交联从小鼠收集的骨骼肌组织，并将组织切碎并超声处理。由于我们发现超速离心不能使用交联肌肉组织将原核与肌原纤维分开，我们设计了一个顺序的过滤程序，以获得没有显着肌原纤维污染的高质量细胞核。我们随后使用超声波仪制备了染色质，并且使用抗BMAL1抗体的ChIP测定显示强大的昼夜节律结合目的基因启动子的BMAL1模式。该过滤方案构成了将高质量细胞核与交联骨骼肌组织隔离的一种易于应用的方法，允许对昼夜节律和其他时间敏感性研究进行一致的样品处理。结合下一代测序（NGS），我们的方法可以部署用于骨骼肌功能的各种机械和基因组研究。

这里我们在组织收集后立即进行甲醛交联，以保持实时的DNA-蛋白质相互作用。然而，我们发现通常用于细胞核分离的蔗糖或胶体梯度23,24在分离细胞核和肌原纤维方面无效（数据未显示）。原因可能是交联赋予核和肌原纤维相似的重力。因此，我们开发了一个串联过滤方法，以有效地从核部分去除大的肌原纤维和其他碎屑。经过100μm过滤后，仍有较大的?...

Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

我们提出了基于过滤的方案，以将高质量的细胞核与交联的小鼠骨骼肌分离，其中我们消除了超离心的需要，使其易于应用。我们显示从核制备的染色质适用于染色质免疫沉淀和可能的染色质免疫沉淀测序研究。

从交联骨骼肌制备高质量核的基于过滤的方法进行染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

a-filtration-based-method-preparing-high-quality-nuclei-from-cross

Research

JoVE Journal

Biochemistry

8.8K 次观看.  University of Texas Health Science Center at Houston. 我们提出了基于过滤的方案，以将高质量的细胞核与交联的小鼠骨骼肌分离，其中我们消除了超离心的需要，使其易于应用。我们显示从核制备的染色质适用于染色质免疫沉淀和可能的染色质免疫沉淀测序研究。 

视频: A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

改进的小鼠骨骼肌染色质沉淀的协议

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

优质发酵鱼产品的制备

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...