PA是奖学金"皮埃尔·杜兰德"的拉瓦尔大学医学院收件人。这项工作是支持由加拿大自然科学和工程研究理事会（MOP-CG095386）马币的多核糖体分馏是通过加拿大创新基金会赠款（MOP-GF091050）所收购，以RMR M拥有一个新的CIHR研究员薪水奖。 我们感谢博士。 E. Khandjian，一Gallouzi，S.迪 - 马可和A. Cammas的有益建议。

该协议遵循的指导方针 经伐的伦理审查委员会。 1，制备细胞培养和脑操作<ol><li>哺乳动物和果蝇细胞<ol><li>生长的HeLa宫颈癌细胞和施奈德果蝇胚胎细胞所推荐的美国典型培养物保藏中心。与细胞在低通道工作。 </li><li>板单元，以达到80％汇合的实验当天。为获得最佳结果，用〜12×10 6个细胞的每个实验条件。在进行提取物为多核糖体分析，刺激翻译，加入新鲜的完全培养基为至少90分钟。 </li></ol></li><li>脑隔离:从年龄出生后9天和16天之间隔离的小鼠大脑。安乐死涉及麻醉和颈椎脱位后的CO 2吸入。下面的牺牲，隔离整个大脑和任何地方，我T AT -80°C或直接将其传送到冷PBS 1X立即使用。 </li></ol> 2，密度的制备梯度分馏系统<ol><li>用0.1％SDS洗管路系统5分钟。 </li><li>用DEPC水洗涤前5分钟，用70％的乙醇5分钟，洗管路系统，然后用RNaseZAP解。 </li><li>泵空气以干燥装置的管道系统。 </li></ol>在蔗糖梯度的3制备<ol><li>使在20mM的Tris-HCl pH 7.4的，1.25毫摩尔MgCl 2，150 mM氯化钠，和1mM DTT的15％和55％的蔗糖溶液。 </li><li>生成 使用ISCO型号160梯度前，所描述的制造商的说明书线性15-55％的蔗糖梯度。然而，重要的是要控制好将TRIS蠕动泵的速度，以避免建立在梯度气泡或湍流。保持梯度在4℃直至使用。 </li><li>在梯度壶程序运行时的时间，冷却超速离心机到4°C。 </li></ol> 4，制备细胞和小鼠脑提取物<ol><li>细胞提取物的制备<ol><li>地方板置于冰上和洗细胞（S）3倍用冷PBS 1X。 </li><li>收获细胞（〜12×10 6）在1ml裂解缓冲液（20mM的Tris-HCl pH为7.4，1.25毫摩尔MgCl 2，150 mM氯化钠，1mM的DTT，1％诺乃洗涤剂P40，5单位/毫升RNA酶抑制剂，补充具有完整的迷你EDTA蛋白酶抑制剂混合片），将它们传输到Eppendorf管中，并通过一个1CC U100胰岛素注射器28 G 1/2通过他们15X拌匀。让细胞裂解物搁置在冰上15分钟。 </li><li>放几滴细胞裂解物上的滑动通过使用10X物镜在相衬显微镜观察，以评估细胞的裂解。只有原子核应该是可见的，并没有细胞膜应该是可见的公证人进行细胞裂解。作为对照，观察未裂解细胞。 </li><li>通过离心澄清的细胞裂解物在11,000×g离心20分钟，在4℃，并保持含有多聚核糖体的可溶性裂解物。 </li></ol></li><li>小鼠脑组织提取物的制备<ol><li>由10个冲程在冰冷的Dounce匀浆2毫升的裂解缓冲液中匀化整个孤立大脑（〜500mg）和通过离心澄清的匀浆液以11,000×g离心15分钟，在4℃下</li><li>加载得到的上清液至50％蔗糖的缓冲垫，并使用超速离心机转子TH-641在4℃下进行沉淀2小时以200,000×g离心</li><li>重悬含在1ml裂解缓冲液中的多聚核糖体的半透明颗粒和通过上下吹打混匀。装载到梯度之前让在冰上30分钟，悬浮液其余部分。 </li></ol></li></ol> 5，加载到提取蔗糖梯度超速离心和<ol><li>测量浓度的RNA存在于使用分光光度计的胞质提取物。小心缓慢地加载〜20 OD 260单位提取物到15％至55％的蔗糖梯度。确保有2-3毫米的可用空间在管的表面，以避免在离心过程中溢流管。 </li><li>使用超离心2小时30分钟，在230000×g离心使用超速离心机转子TH-641在4℃下离心梯度当离心结束后，取出试管，并将其放置在冰小心不要打扰梯度。 </li></ol>细胞质提取物6。分级为多聚核糖体剖析<ol><li>将蔗糖梯度的密度自动分级系统，并进行分数的自动分离和收集（每次0.5毫升），所描述的制造商。 </li><li>收集各馏分（〜500μl）入个人Eppendorf管在254 nm处连续监测吸光度。在parall梯度分馏操作的EL中，polyribosomal配置文件将被编辑在记录纸上。或者，使用附加到图表记录器有一个电子采集的多核糖体轮廓的数据采集单元。 </li><li>在每次运行结束后，转移干冰收集Eppendorfs。此时，无论是存储收集的馏分在-80℃或直接沉淀蛋白质-RNA复合物，如下所示。 </li></ol> 7，蛋白提取及分析<ol><li>蔗糖梯度的每个收集到的馏分，加入2倍体积的100％冷乙醇，并让RNA-蛋白复合物沉淀在-20℃下过夜。 </li><li>离心每个RNA-蛋白沉淀于11000×g离心20分钟，在4℃下然后用70％的乙醇。干燥并在继续进行Western印迹使用特异性抗体（参见图2）之前悬浮在SDS-PAGE样品缓冲液中的沉淀物。 </li></ol> 8，RNA提取和Analysis <ol><li>沉淀RNA-蛋白质复合物在7.1节。每个重悬RNA-蛋白质沉淀中含有0.1％SDS裂解缓冲液。消化与2在55℃水浴中30分钟毫克/毫升蛋白酶K的析出物的蛋白质组分。 </li><li> ":氯仿酚"，2倍体积的氯仿和0.1体积的2M醋酸钠pH值为4，以10000×g离心，在4℃下进行20分钟，加入1倍体积的提取沉淀的RNA元件。沉淀的RNA从每个样品中加入1倍体积的异丙醇和0.2微克/微升糖原的所得水相过夜，在-20℃下。旋转沉淀1小时以10000×g在4℃下用70％冷乙醇洗涤RNA沉淀。 </li><li>重悬RNA沉淀成小体积的无RNA酶的水。评估的数量 和质量 RNA的使用分光光度计。 </li></ol>

<ol>
	<li>Gebauer, F., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+translational+control.">Molecular mechanisms of translational control.</a> Nature Reviews. Molecular Cell Biology. 5 (10), 827-835 (2004).</li><li>Jackson, R. J., Hellen, C. U. T., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nature Reviews. Molecular Cell Biology. 11 (2), 113-127 (2010).</li><li>Fournier, M. -. J., Gareau, C., Mazroui, 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+chemotherapeutic+agent+bortezomib+induces+the+formation+of+stress+granules.">The chemotherapeutic agent bortezomib induces the formation of stress granules.</a> Cancer Cell International. 10 (12), (2010).</li><li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nature Reviews. Molecular Cell Biology. 6 (4), 318-327 (2005).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Filion, C., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+of+messenger+RNA+by+Fragile+X+Mental+Retardation+protein+into+cytoplasmic+granules+induces+translation+repression.">Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.</a> Human Molecular Genetics. 11 (24), 3007-3017 (2002).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Boilard, N., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+Mental+Retardation+protein+determinants+required+for+its+association+with+polyribosomal+mRNPs.">Fragile X Mental Retardation protein determinants required for its association with polyribosomal mRNPs.</a> Human Molecular Genetics. 12 (23), 3087-3096 (2003).</li><li>Farny, N. G., Kedersha, N. L., Silver, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Metazoan+stress+granule+assembly+is+mediated+by+P-eIF2alpha-dependent+and+-independent+mechanisms.">a Metazoan stress granule assembly is mediated by P-eIF2alpha-dependent and -independent mechanisms.</a> RNA. 15 (10), 1814-1821 (2009).</li><li>Gareau, C., Houssin, E., 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=Characterization+of+fragile+x+mental+retardation+protein+recruitment+and+dynamics+in+Drosophila+stress+granules.">Characterization of fragile x mental retardation protein recruitment and dynamics in Drosophila stress granules.</a> PLoS ONE. 8 (2), (2013).</li><li>Brackett, D. M., Qing, F., Amieux, P. S., Sellers, D. L., Horner, P. J., Morris, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FMR1+transcript+isoforms:+association+with+polyribosomes;+regional+and+developmental+expression+in+mouse+brain.">FMR1 transcript isoforms: association with polyribosomes; regional and developmental expression in mouse brain.</a> PLoS ONE. 8 (3), (2013).</li><li>Khandjian, E. W., Huot, M. -. E., Tremblay, S., Davidovic, L., Mazroui, R., Bardoni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+evidence+for+the+association+of+fragile+X+mental+retardation+protein+with+brain+polyribosomal+ribonucleoparticles.">Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (36), 13357-13362 (2004).</li><li>Erikson, A., Winblad, B., Wallace, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+gene+expression+in+the+human+brain.">Translational control of gene expression in the human brain.</a> Prog Neuropsychopharmacol Biol Psychiatry. 13 (3-4), 469-479 (1989).</li><li>Stephens, S. B., Nicchitta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V+In+vitro+and+tissue+culture+methods+for+analysis+of+translation+initiation+on+the+endoplasmic+reticulum.">V In vitro and tissue culture methods for analysis of translation initiation on the endoplasmic reticulum.</a> Methods in Enzymology. 431, 47-60 (2007).</li><li>Koritzinsky, M., Wouters, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+regulation+of+messenger+RNA+translation.">Hypoxia and regulation of messenger RNA translation.</a> Methods in Enzymology. 435, 247-273 (2007).</li><li>Khandjian, E. W., Corbin, F., Woerly, S., Rousseau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fragile+X+mental+retardation+protein+is+associated+with+ribosomes.">The fragile X mental retardation protein is associated with ribosomes.</a> Nature Genetics. 12, 91-93 (1996).</li><li>Sivan, G., Kedersha, N., Elroy-Stein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+slowdown+mediates+translational+arrest+during+cellular+division.">Ribosomal slowdown mediates translational arrest during cellular division.</a> Molecular and Cellular Biology. 27 (19), 6639-6646 (2007).</li><li>Thomas, J. D., Johannes, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mRNAs+that+continue+to+associate+with+polysomes+during+hypoxia.">Identification of mRNAs that continue to associate with polysomes during hypoxia.</a> RNA. 13, 1116-1131 (2007).</li><li>Kumaraswamy, S., Chinnaiyan, P., Shankavaram, U. T., L&#252;, X., Camphausen, K., Tofilon, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+gene+translation+profiles+reveal+tumor+type+and+cancer-specific+components.">Radiation-induced gene translation profiles reveal tumor type and cancer-specific components.</a> Cancer Research. 68 (10), 3819-3826 (2008).</li><li>Fournier, M. -. J., Coudert, L., 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=Inactivation+of+the+mTORC1-eIF4E+Pathway+alters+Stress+Granules+Formation.">Inactivation of the mTORC1-eIF4E Pathway alters Stress Granules Formation.</a> Molecular and Cellular Biology. 33 (11), 2285-2301 (2013).</li><li>Sanchez, G., Dury, A. Y., 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=A+novel+function+for+the+survival+motoneuron+protein+as+a+translational+regulator.">A novel function for the survival motoneuron protein as a translational regulator.</a> Human Molecular Genetics. 22 (4), 668-684 (2013).</li><li>B&#233;chade, C., Rostaing, P., 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=Subcellular+distribution+of+survival+motor+neuron+(SMN)+protein:+possible+involvement+in+nucleocytoplasmic+and+dendritic+transport.">Subcellular distribution of survival motor neuron (SMN) protein: possible involvement in nucleocytoplasmic and dendritic transport.</a> The European Journal of Neuroscience. 11 (1), 293-304 (1999).</li><li>Goulet, I., Boisvenue, S., Mokas, S., Mazroui, R., C&#244;t&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDRD3,+a+novel+Tudor+domain-containing+protein,+localizes+to+cytoplasmic+stress+granules.">TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules.</a> Human Molecular Genetics. 17 (19), 3055-3074 (2008).</li><li>Nottrott, S., Simard, M. J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+let-7a+miRNA+blocks+protein+production+on+actively+translating+polyribosomes.">Human let-7a miRNA blocks protein production on actively translating polyribosomes.</a> Nature Structural &amp; Molecular Biology. 13 (12), 1108-1114 (2006).</li><li>Genolet, R., Araud, T., Maillard, L., Jaquier-Gubler, P., Curran, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+analyse+the+specific+impact+of+rapamycin+on+mRNA-ribosome+association.">An approach to analyse the specific impact of rapamycin on mRNA-ribosome association.</a> BMC Medical Genomics. 1 (33), (2008).</li><li>Del Prete, M. J., Vernal, R., Dolznig, H., M&#252;llner, E. W., Garcia-Sanz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Isolation+of+polysome-bound+mRNA+from+solid+tissues+amenable+for+RT-PCR+and+profiling+experiments.">a Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.</a> RNA. 13 (3), 414-421 (2007).</li><li>Thoreen, C. C., Chantranupong, L., Keys, H. R., Wang, T., Gray, N. S., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unifying+model+for+mTORC1-mediated+regulation+of+mRNA+translation.">A unifying model for mTORC1-mediated regulation of mRNA translation.</a> Nature. 485 (7396), 109-113 (2012).</li><li>Ingolia, N. T., Brar, G. A., Rouskin, S., Mcgeachy, A. M., Weissman, 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=The+ribosome+profiling+strategy+for+monitoring+translation+in+vivo+by+deep+sequencing+of+ribosome-protected+mRNA+fragments.">The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.</a> Nature Protocols. 7 (8), 1534-1550 (2012).</li><li>Morris, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+footprints+on+a+transcriptome+landscape.">Ribosomal footprints on a transcriptome landscape.</a> Genome Biology. 10 (4), (2009).</li></ol>

作者什么都没有透露。

在蔗糖梯度上的多核糖体轮廓分析允许翻译起始的测量通过分析多核糖体从细胞或组织9,11-14孤立的密度。这种技术是最好的（如果不是唯一的）方法来衡量翻译起始体内 。它是用来监视在细胞周期15生长的细胞的平移状态，并评估各种类型的应力，包括病毒感染，缺氧13,16，辐射17，和化学药物治疗中使用的18的作用，对翻译起始。然而，尽管这种技术能够很好地评估细胞培养物和特定的组织如脑和肝的翻译起始，它仍然是，如果它可以被用来比较的正常翻译起始与患病的组织和肿瘤中建立的。 也可以说改变翻译elonga条件下发生变化，多聚核糖体型材化或终止。在这些情况下，翻译的伸长率的抑制将导致增加的多核糖体的峰而终止的抑制应该导致相对于对照更大的多核糖体。相比之下，翻译起始的抑制剂防止核糖体的改造，因为形成积极的翻译起始复合物的被封锁。这是通过与80S monosomes随之增加减少的多核糖体的峰反映在多聚核糖体访问。因为翻译抑制剂搪塞翻译起始复合物，因此预计这两个40S和60S高峰也将增加。可是，很多时候增加只有80S monosomes是在抑制翻译起始的观察。为什么增加另一个核糖体复合物是不遵守的原因目前还不清楚。一个可能的解释可能是提取导致观察到的积累过程中可能出现停滞核糖体复合物之间的偶然协会化的只有80S monosomes。 除了翻译起始的分析，核糖体配置技术可以帮助确认翻译起始因子（如eIF4E的，eIF4A和eIF4G）的身份，并确定新的翻译稳压器。作为翻译起始因子被释放从核糖体上一次翻译起始步骤，它们不应该被过度翻译的核糖体组分进行检测。蛋白质如FMRP 14，SMN 19,20，TDRD3 21与两个多聚核糖体和非核糖体组分的关联表明这些蛋白在翻译的调控作用并不限于起始步骤。监测与多聚核糖体蛋白的协会也构成了强有力的手段来评估应力条件如何影响特定蛋白质的翻译活动。然而，特定的蛋白质与多聚核糖体本身的关联是不足以得出结论平移ROLE，然后需要通过功能研究证实。最后，将多核糖更新技术允许确定在各种条件下特异mRNAs的翻译活性，并进行检查的调控翻译如的miRNA介导的翻译抑制22特定的机制。一般使用多核糖体的分析可以进一步延长使用各种下游应用，包括全基因组微阵列分析，以及最近的转录组尺度核糖体分析23-26允许蛋白表达和更重要的是提供一种新的强有力的实验工具的鲁棒预测平移控制23-27的分析。 正如任何方法，注意事项需要同时执行多核糖体分析才能作出。在这方面，多个参数，例如孔通道，细胞裂解，细胞提取物被装载到该梯度量和RNA完整性应该是C井ontrolled。还需要以超离心条件的优化，得到核糖体人口通过蔗糖梯度上最好分离。构成该梯度和裂解缓冲液，例如蔗糖和洗涤剂组分，可在254纳米波长的吸光度得到。因此，这是重要的，包括只含有裂解缓冲液的控制梯度。最后，梯度本身应谨慎处理，因为梯度极小扰动可引起多聚核糖体型材巨大的影响。

真核细胞中不断遇到各种需要快速适应性细胞反应有害的生理和环境应激条件。细胞应激反应涉及抗生存和促存活作用因子之间的精确平衡。破坏这种平衡可以有不可逆的后果导致人类疾病如癌症和神经退行性疾病的发展。在应激反应的第一步骤中，细胞激活，涉及改变基因表达在mRNA翻译水平的协调控制促存活途径。 在真核生物mRNA的翻译是涉及翻译起始因子（EIFS），特定的RNA结合蛋白（其中，RBD）和RNA分子1之间协调的相互作用复杂的细胞过程。 mRNA翻译被分成三个不同的阶段:起始，延伸和终止。虽然所有三个阶段都受到REGUlatory机制，平移控制机制，针对翻译的大多是起始阶段，这足以构成蛋白质的合成2的限速步骤。 翻译起始是一个高度有序的过程，开始于eIF2a.GTP.Met-tRNA的形成我遇到了三元复合物及其随后的结合到40S核糖体亚单位，从而导致预起始复合物的形成。接下来的步骤是将起始前复合物的mRNA，它涉及到翻译起始因子如eIF4F和eIF3的活性的募集。由此形成的48S起始前复合物经受特定的构象变化，使此机器开始扫描mRNA的5&#39;-非翻译区，直到它识别起始密码子八月大部分的翻译起始因子，然后释放，60S亚基招募形成80S核糖体复合胜任翻译，一吨点蛋白质合成开始（ 图1），其中。多个80S monosome可翻译的mRNA相同的时间产生所谓的多聚核糖体（或者多聚核糖体）。多聚核糖体上的mRNA的密度反映了起始，延长和终止率，因而是一种特殊的转录物的可译性的度量。然而，多核糖更新主要用于在启动步骤，以评估变化的mRNA的翻译。在这里，我们使用了蛋白酶体抑制剂作为翻译起始抑制剂。癌细胞与这种药物治疗引起应激反应特点是激活的磷酸化翻译起始因子eIF2a 3名为HRI应力激酶。 eIF2a的磷酸化是重要的事件，导致翻译起始的在哺乳动物细胞中4的抑制作用1。

<table><tbody><tr><td colspan="4">Cells</td></tr><tr><td>HeLa cervical cancer cells</td><td>American Type Culture Collection (Manassas, VA; ATCC)</td><td>CCL-2</td><td></td></tr><tr><td>Schneider &lt;em&gt;Drosophila&lt;/em&gt; embryonic cells</td><td>American Type Culture Collection (Manassas, VA; ATCC)</td><td>CRL-1963</td><td></td></tr><tr><td colspan="4">Culture medium and Supplements</td></tr><tr><td>Schneider&amp;rsquo;s &lt;em&gt;Drosophila&lt;/em&gt; Medium</td><td>Sigma-Aldrich</td><td>SO146-500ml</td><td>&amp;nbsp;</td></tr><tr><td>DMEM</td><td>Life technologies</td><td>11995-073</td><td>&amp;nbsp;</td></tr><tr><td>FBS</td><td>Fisher Scientist Scientist</td><td>SH30396-03</td><td>&amp;nbsp;</td></tr><tr><td>Penicillin/streptomycin</td><td>Life technologies</td><td>15140122</td><td></td></tr><tr><td colspan="4">Sucrose solutions</td></tr><tr><td>D-sucrose</td><td>Fisher Scientist</td><td>BP220-212</td><td>&amp;nbsp;</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>49767</td><td>&amp;nbsp;</td></tr><tr><td>Bromophenol blue</td><td>Fisher Scientist</td><td>B3925</td><td></td></tr><tr><td colspan="4">Lysis buffer</td></tr><tr><td>Tris HCl</td><td>Fisher Scientist</td><td>BP153-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2670-100G</td><td></td></tr><tr><td>NaCl</td><td>Tekniscience</td><td>3624-05</td><td>&amp;nbsp;</td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D 9779</td><td>&amp;nbsp;</td></tr><tr><td>Nonidet P40 (Igepal CA-630 )</td><td>MJS Biolynx</td><td>19628</td><td>&amp;nbsp;</td></tr><tr><td>SDS</td><td>Tekniscience</td><td>4095-02</td><td>&amp;nbsp;</td></tr><tr><td>RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td><td>Life technologies</td><td>10777-019</td><td>&amp;nbsp;</td></tr><tr><td>Antiproteases (complete, mini, EDTA free)</td><td>Roche</td><td>11,836,170,001</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">RNA Extraction</td></tr><tr><td>Proteinase K</td><td>Life technologies</td><td>AM2542</td><td>&amp;nbsp;</td></tr><tr><td>Phenol:chloroform</td><td>Fisher Scientist</td><td>BP1754I-400</td><td>&amp;nbsp;</td></tr><tr><td>Chloroform</td><td>Fisher Scientist</td><td>C298-500</td><td>&amp;nbsp;</td></tr><tr><td>Glycogen</td><td>Life technologies</td><td>10814-010</td><td>&amp;nbsp;</td></tr><tr><td>Isopropanol</td><td>Acros organics</td><td>327270010</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">Antibodies</td></tr><tr><td>anti-FMRP antibody</td><td>Fournier &lt;i&gt;et al.&lt;/i&gt;, Cancer Cell International, 2010</td><td></td><td>&amp;nbsp;</td></tr></tbody></table>

analysis of translation initiation during stress conditions by polysome profiling

mRNA翻译的精确控制是基本的真核细胞内环境稳定，特别是在应对生理和病理的压力。这个方案的改变可导致受损细胞的生长，癌症发展的一个标志，或过早的细胞死亡如见于神经变性疾病。多的关于对翻译控制的分子基础所谓已经从多核糖体分析使用密度梯度分级分离系统获得的。这种技术依赖于细胞质提取物对超速的线性蔗糖梯度。一旦旋转完成时，该系统允许分馏对应于不同翻译离心分离区与定量核糖体种群，从而导致多核糖体模式。变化多核糖体轮廓是指示发生在响应各类应力的翻译起始的变化或缺陷。此技术还允许以评估日特定蛋白对翻译起始é角色，并衡量特定mRNA的翻译活动。这里我们描述的协议，以便评估在正常或胁迫生长条件下的真核细胞和组织的翻译起始执行多核糖访问。

如前面提到的，多核糖体轮廓允许翻译起始的胁迫条件下的变化的分析， 图1是如前面所述是一个涉及翻译起始复合物的有序组装多步骤的过程翻译起始的简化视图。在正常生长条件下，翻译起始复合物被转化为多核糖体，其检测由多核糖更新证实为一个活跃的翻译起始（ 图2;未处理）。根据应力条件然而，翻译起始被阻断导致80S monosomes积累和减少的多核糖体的峰（ 图2;蛋白酶体抑制剂）。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51164/51164fig1highres.jpg" src="/files/ftp_upload/51164/51164fig1.jpg" width="500px" /> 图1。一个S<html>翻译起始implified看法。 &#160;翻译起始包括几个步骤:1）eIF2a.GTP.Met酰-tRNA的形成， 我遇到了三元复合物，2）的三元复合物与40S的核糖体的关联形成43S起始前复杂，43S复合物与mRNA的3）的关联形成48S起始前复合物，4）扫描所述mRNA的5&#39;-非翻译区由48S核糖体，直到它到达与识别起始AUG密码，以及5）招募的核糖体大亚基60S到48S形成在该点的80S复杂多肽合成开始。翻译起始的每一步都涉及到几个翻译起始因子的活性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51164/51164fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 upload/51164/51164fig2highres.jpg"SRC ="/ files/ftp_upload/51164/51164fig2.jpg"宽度="500px的"/&gt; 图2的多核糖体访问和多核糖体的分析通过在HeLa细胞蛋白质印迹相关蛋白 （A - B）。细胞质提取物从无论是在正常条件（A）或在与蛋白酶体抑制剂为4小时（B治疗生长的HeLa细胞制备的），并离心以15-55％V / W线性蔗糖梯度。多聚核糖体配置文件显示在顶部面板。的40S核糖体，核糖体60S，80S monosomes和多聚核糖体的位置显示在每个配置文件。馏分从顶部（15％）的梯度的底部（55％）显示由左到右。收集级份，并通过蛋白印迹分析（底部小图）进行分析以抗核糖体大L28蛋白以及与多核糖体相关蛋白FMRP是作为核糖体完整性5,6的控制。需要注意的是TREatment与蛋白酶体抑制剂可减少多核糖体的峰与伴随增加的80S monosomes指示翻译起始的抑制。施耐德果蝇细胞或小鼠大脑获得的典型配置文件已在别处7-10描述。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51164/51164fig2highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

在这里，我们描述一个变化来分析真核细胞中的mRNA的翻译的响应于胁迫条件的启动方法。此方法是基于换算从非翻译的核糖体的核糖体的蔗糖梯度的速度分离。

翻译起始的分析在应力条件由多聚核糖体剖析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

analysis-translation-initiation-during-stress-conditions-polysome

Nanyang Technological University

Research

JoVE Journal

Biology

18.2K 次观看.  Laval University. 在这里，我们描述一个变化来分析真核细胞中的mRNA的翻译的响应于胁迫条件的启动方法。此方法是基于换算从非翻译的核糖体的核糖体的蔗糖梯度的速度分离。 

视频: Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

生物学

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

使用多体分析在发育中的鼠标大脑中进行翻译分析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

神经科学

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

植物多聚核糖体剖面的简单方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

翻译起始的分析在应力条件由多聚核糖体剖析

摘要

探索更多视频

此视频中的章节

核糖体剖面对芽殖酵母中翻译的全基因组定量

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

无需梯度测定器或分馏系统的多聚体分析

真核Polyribosome剖面分析

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

使用多体分析在发育中的鼠标大脑中进行翻译分析

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

植物多聚核糖体剖面的简单方法

从真核细胞中快速体内固定和分离转化复合物

翻译起始的分析在应力条件由多聚核糖体剖析

摘要

探索更多视频

此视频中的章节

核糖体剖面对芽殖酵母中翻译的全基因组定量

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

无需梯度测定器或分馏系统的多聚体分析

真核Polyribosome剖面分析

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

使用多体分析在发育中的鼠标大脑中进行翻译分析

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

植物多聚核糖体剖面的简单方法

从真核细胞 中快速体内 固定和分离转化复合物

从真核细胞中快速体内固定和分离转化复合物