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>

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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?...

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。

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	</colgroup>
	<tbody>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
			<td height="21" style="height:21px;width:279px;">Cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">HeLa cervical cancer cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Schneider Drosophila embryonic cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CRL-1963</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Schneider&#8217;s Drosophila Medium</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>SO146-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM</td>
			<td>Life technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">FBS</td>
			<td>Fisher Scientist Scientist</td>
			<td>SH30396-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">penicillin/streptomycin</td>
			<td>Life technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Sucrose solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">D-Sucrose</td>
			<td>Fisher Scientist</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Blue Bromophenol</td>
			<td>Fisher Scientist</td>
			<td>B3925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tris Hydrochloride</td>
			<td>Fisher Scientist</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:279px;">MgCl2 </td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NaCl</td>
			<td>Tekniscience</td>
			<td>3624-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DTT</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D 9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nonidet P40 (Igepal CA-630 )</td>
			<td>MJS Biolynx</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SDS</td>
			<td>Tekniscience</td>
			<td>4095-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td>
			<td>Life technologies</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Antiproteases (complete, mini, EDTA free)</td>
			<td>Roche</td>
			<td>11,836,170,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">RNA Extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteinase K</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenol: Chloroforme</td>
			<td>Fisher Scientist</td>
			<td style="width:119px;">BP1754I-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Chloroforme</td>
			<td style="width:311px;">Fisher Scientist</td>
			<td style="width:119px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycogen</td>
			<td>Life technologies</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isopropanol</td>
			<td style="width:311px;">Acros organics</td>
			<td>327270010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-FMRP antibody</td>
			<td colspan="2" style="width:429px;">Fournier et al., Cancer Cell International, 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-Ribosomal Protein L28 antibody</td>
			<td style="width:311px;">Santa Cruz Biotechnology, Inc.</td>
			<td style="width:119px;">SC-50362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Others</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteasome inhibitor : Bortezomib</td>
			<td style="width:311px;">LC Laboratories</td>
			<td style="width:119px;">B-1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DEPC (Diethylpyrocarbonate)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D5758-25ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RNaseZAP Solution</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">T25 cell culture flask</td>
			<td style="width:311px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1cc U100 Insulin Syringe 28 G1/2</td>
			<td>Fisher Scientist</td>
			<td>148291B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube ultra-centrifugation, PA, 12ml</td>
			<td>Fisher Scientist</td>
			<td>FSSP9763205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco Model 160 gradient former</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Sorvall OTD Combi</td>
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Thermo Scientific Sorvall Rotor TH-641</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;">54295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Automated Density Fractionation System</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;">67-9000-177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco UA-6 UV-vis detector</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ultracentrifuge C 5415</td>
			<td style="width:311px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Optical Microscope</td>
			<td style="width:311px;">Olympus CK2</td>
			<td style="width:119px;"></td>
			<td></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（ <stro...

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

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

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

植物激素在应激反应监测

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

科研

生物学

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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. ...

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 ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

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 ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...