这项研究得到德意志 Forschungsgemeinschaft (DFG) 在 IRTG 1874 "糖尿病微血管并发症" 和 CRC 1118 "反应性代谢物作为糖尿病晚期并发症的原因" 的支持。N2 和 CL2166 的线虫菌株由 CGC 提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 资助。

注: 本协议分为五节。在1–3节中, 提出了大规模养殖C 型线虫的主要协议。4和5节为评估糖尿病代谢产物中出现的代谢产物提供了附加的协议。详细来说, 第1节描述了一个普通的大范围的板块文化。2节重点介绍了大量的C 线虫的转移, 而3节则解释了大规模样品的收获。4节解释了从样品中分离出的蛋白质, 5 节描述了随后的液相色谱-串联质谱 (LC-ms) 分析的样品制备方法。
1. 板材上的大范围文化
<ol><li>一般情况下, 工作在无菌罩下或在明火旁边以避免污染。</li>
	<li>
要培养蠕虫直到它们到达年轻的成人阶段, 请按照以前发布的7号协议进行以下修改。
	<ol>
<li>使用活大肠杆菌 OP50 细菌喂养线虫, 并准备400µM fluorodeoxyuridine 琼脂 (不氨苄西林/FUdR) 进行实验。</li>
		<li>为同步人群的准备, 使用非浓缩 OP50, 并为成功的实验与成人, 使用3.3x 浓缩 OP50 通过去除70% 的上清。</li>
		<li>建议每日喂食间隔。在评估氧化应激时, 不同的进食间隔和容积可能会影响结果。</li>
		<li>为接种, 采取一个板材与几个线虫并且切开它成片断大约 0.5 x 1 cm2与手术刀。将两到三块转移到线虫生长介质 (NGM) 板上, 直径为60毫米, 含非浓缩 OP50。</li>
		<li>在适宜于应变的温度下孵化板材 (野生型 N2 应在20摄氏度培养), 直到鸡蛋在盘子上可见。</li>
		<li>用2毫升的 M9 缓冲液冲洗掉大部分的卵和成年动物, 并在15毫升管中收集它们。离心机悬浮在 800 x g为2分钟。同时, 开始准备漂白液。</li>
		<li>取出离心管, 用10毫升吸管取出 M9 缓冲器。添加10毫升的漂白溶液和混合在漩涡, 直到成人线虫裂解。这通常采取大约 5–7 min, 但不应该花费超过15分钟或蛋不会完全地以后开发在实验中。</li>
		<li>离心机悬浮在 800 x g为2分钟, 洗3次与10毫升 M9 缓冲, 以清除鸡蛋从漂白溶液。</li>
		<li>现在添加150µL 的蛋悬浮在 OP50 板上。每个盘子上都要有200–300xg 蛋的密度。等到线虫到达成年阶段。 注: 所述实验所用的 M9 缓冲器 (pH 7.0) 由以下物质组成:22 毫米,2PO4, 42 毫米 Na2HPO4, 和86毫米氯化钠。热处理后加入1毫米 MgSO4。漂白溶液含有0.5 米氢氧化钠, 2.3 毫米 NaOCl 在蒸馏水中解决。</li>
		<li>准备无菌 M9 缓冲剂, 不育的吸管提示用切尖冲洗蠕虫, 和15毫升管收集他们在。</li>
		<li>在盘子上涂上大约1毫升的 M9 缓冲器, 轻轻地将吸管的线虫放在盘子上, 轻柔地散发缓冲器。</li>
		<li>现在, 收集管中的线虫。直接应用150µL 悬浮在板材 (种子与 OP50) 与切开的吸管提示, 并且显微镜下评估线虫的密度 (推荐: 50–100线虫每60毫米板材)。</li>
		<li>如果它们过于致密, 稀释悬浮与 M9 缓冲, 并评估它在显微镜下, 直到达到预期的密度。如果产量太少, 让蠕虫安定下来或离心机悬浮十年代在 800 x g去除液体。 注: 一个充分的密度的例子在图 1A (宏观) 和1B (微观) 在20X 增量给。按照所描述的经验进行。</li>
		<li>请记住, 蠕虫很快就会安定下来。为了保证板材之间的均匀分布, 在每叠板 (第4-5 板) 后反转管。 注: 切割吸管尖端, 以及温和摆动和洗涤的盘子, 减少剪应力。</li>
	</ol>
</li>
</ol>2.大量秀丽线虫的转移
<ol><li>用大约500µL 的 M9 缓冲剂洗涤线虫, 这取决于盘子的年龄和干燥程度。使用相同的缓冲, 重复分离线虫, 直到大多数动物被收集在悬浮。</li>
	<li>慢慢地用割下的吸管把线虫带上, 并将它们转移到 OP50 准备好的盘子上。让盘子干。 注意: 注意不要比推荐的卷应用得多。特别是在长期的实验中, 板块可能无法耐受, 琼脂也会断裂, 使线虫爬入裂缝中。</li>
</ol>3. 大秀丽线虫样品的收获
<ol><li>根据所选的方法, 开始实验, 有足够数量的C. 线虫。对于 LC-质谱/ms 分析, 每组20板 (60 毫米 x 15 毫米), 每片约100个线虫, 以确保每样蛋白质至少有200µg 的产量。 注: 本议定书的这一部分不再需要在无菌条件下工作, 因为线虫将被收集和冻结。</li>
	<li>在样品收集的当天, 准备液氮以对收集的样品进行冻结。在冰上保持无菌 M9 缓冲, 以减缓线虫的新陈代谢。</li>
	<li>用1毫升的 M9 缓冲器, 轻轻地旋转盘子, 从盘子里洗去线虫。这避免收集已故的线虫和多数蛋, 如果有任何礼物, 因为他们坚持细菌和琼脂。</li>
	<li>把音量调到15毫升管上。</li>
	<li>收集完样品后, 等待线虫在 800 x g处简单地沉降或离心管, 并取出大部分 M9 缓冲器。用大约10毫升的 M9 (10x 样本量) 冲洗样品3x 以除去任何细菌。 注: 为确保所有已死亡的线虫被除去, 蔗糖浮选是另一种选择。然而, 这是不适合的显示实验15。蔗糖是水解葡萄糖和果糖。在所描述的实验中, 评价葡萄糖的作用, 以促进慢性糖尿病的生化变化。因此, 蔗糖浮选可能会混淆结果。</li>
	<li>准备一个1.5 毫升管与1毫升的 M9 缓冲器和一个空1.5 毫升管每样。将球团与玻璃吸管从15毫升管转移到空1.5 毫升管。这一步骤对于确保定量转移至关重要。 注意: 不像以前的步骤, 这里的线虫更集中。由于可能坚持塑料吸管的提示, 损失的大部分样品是预期的。因此, 用玻璃吸管代替。</li>
	<li>转移后, 使用玻璃吸管从第二个1.5 毫升管中拿起1毫升的 M9 缓冲器, 从吸管壁上冲洗线虫。另外, 从15毫升管中清洗剩余的线虫, 并将其转移到样品管。</li>
	<li>离心样品管在 1.9万 x g 1 分钟, 然后删除尽可能多的上清。</li>
	<li>用 Parafilm 封住管子, 并立即将其冻结在液氮中。贮存管在-80 摄氏度, 直到裂解制剂。</li>
</ol>4. 质谱分离蛋白
注意: 这里描述的蛋白质分离也可以用于其他化验 (例如, 西方印迹)。
<ol><li>对冷冻样品, 添加相同数量的0.5 毫米氧化锆珠和至少2x 的裂解液容量含有蛋白酶抑制剂鸡尾酒。 注: 用100µL 缓冲剂进行了描述的线虫-蛋白质相关分析。裂解200µL 的缓冲液。</li>
	<li>在速度9上启动1分钟的均质机。确保样品完全裂解。如果没有完全裂解, 请重复此步骤。</li>
	<li>离心样品30分钟在4°c 在 1.9万 x g。将上清液转移到冰上的新管上, 将其储存在-80 摄氏度, 直到进一步测量。 注: 所述实验所用的非变性缓冲器由以下物质组成:20 毫米三盐酸 (pH 8)、137毫米氯化钠、1% 海卫 x 和2毫米 EDTA。 注: 遵循本议定书的步骤, 并应用了建议的规模, 蛋白质颗粒从大约1000线虫的产量应约500µg。有关更多比较, 请参阅图 2和代表结果。</li>
</ol>5. 液相色谱-串联质谱测量样品的制备
注: 根据感兴趣的参数, 样品准备会有所不同。本议定书的重点是丙酮醛和年龄确定。
<ol><li>
用 12-diaminobenzene (DB) 的衍生化方法测量胞内丙酮醛, 如前所述9。
	<ol>
<li>融汇样品与20µL 冰冷 20% (重量/卷) 乙酸酸在 0.9% (重量/卷) 氯化钠和80µL 水在1.5 毫升管子。</li>
		<li>用内部标准 (13C3毫克; 400 nM) 将5µL 的样品钉在一起, 然后用涡流将它们混合, 然后在 2.1万 x g处将其离心为5摄氏度。</li>
		<li>将上清的35µL 转移到含有200µL 玻璃插入物的 HPLC 样品瓶中。</li>
		<li>将5µL 3% 叠氮化钠 (重量/卷) 添加到每个样品, 后跟10µL 0.5 毫米 DB 在200毫米 HCl 中含有0.5 毫米 diethylenetriaminepentaacetic 酸 (DETAPAC) 在水中。</li>
		<li>在室温下孵化4小时的样品, 免受光照。</li>
		<li>如前文所述, 分析样品的 LC-ms/毫秒,9。 注: 对于年龄的测量, 请将该协议第4节所述的蛋白质分离出来。</li>
	</ol>
</li>
	<li>
用布拉德福德10测定 (解冻) 裂解物的蛋白质浓度。
	<ol>
<li>在磷酸盐缓冲盐水 (PBS) 中, 用牛血清白蛋白 (BSA) 解决的标准曲线整除数30µL 的含量: 0;0.1;0.2;0.3;0.4;0.6;0.8;和1.0 毫克/dL。 注: 如果样品是按照建议的尺寸制备的 (见步骤 4.1, 用于制备具有200µL 缓冲液的裂解剂), 估计浓度应在大约2–7毫克/毫升范围内。在这种情况下, 在测量之前用 PBS 稀释样品1:10。 注意: 对于第一个使用此方法的实验, 建议对不同稀释 (1、1:10 和 1:100) 中的样品进行测量。由于没有确切的确定线虫数量, 不同的研究人员的产量可能有所不同。</li>
		<li>使用透明的96井板 (聚苯乙烯) 和吸管10µL 的每个标准浓度和样品在选定的稀释中重复的水井。</li>
		<li>用蒸馏水 (蒽醌) 稀释蛋白质染料试剂浓缩1:5。工作台)并添加200µL 的稀释到每个样品上的盘子。在室温下孵育10分钟的盘子。</li>
		<li>测量30分钟内的吸光度在595毫微米与一个板块阅读器。样品可以从计算的标准曲线中插值。该方法的详细资料在文献10中得到了广泛的描述。</li>
	</ol>
</li>
	<li>
测量细胞内的年龄, 如前所述11,12。
	<ol>
<li>在 10 kDa 离心过滤装置中, 用离心清洗总蛋白提取物 (约200µg) 3x。</li>
		<li>加入10µL 100 毫米盐酸, 10 µL 胃蛋白酶 (2 毫克/毫升在20毫米 hcl) 和10µL 麝香 (2 毫克/毫升在20毫米 hcl) 到恢复的蛋白质 (大约100µL) 和孵化样品在37°c 为 24 h。</li>
		<li>通过添加12.5 µL 0.5 米磷酸钾缓冲液 (ph 7.4) 和5µL 260 毫米的 KOH, 中和并缓冲样品在 pH 值7.4。</li>
		<li>添加10µL 链霉蛋白酶 E [2 毫克/毫升10毫米磷酸钾缓冲液 (pH 7.4)] 和10µL 青霉素-链霉素溶液, 并孵化样品在37°c 24 h。</li>
		<li>添加10µL 的氨基 [2 毫克/毫升10毫米磷酸钾缓冲 (ph 7.4)] 和10µL 脯肽酶 [2 毫克/毫升在10毫米磷酸钾缓冲 (pH 7.4)], 并孵化样品在37°c 48 h。</li>
		<li>分析所产生的水解物的 LC-ms/毫秒, 如前所述12。</li>
	</ol>
</li>
</ol>

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(64), e4019 (2012).</li><li>Lagido, C., McLaggan, D., Glover, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screenable+In+Vivo+Assay+for+Mitochondrial+Modulators+Using+Transgenic+Bioluminescent+Caenorhabditis+elegans.">A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.</a> Journal of Visualized Experiments. (105), e53083 (2015).</li><li>Takamiya, S., Mita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+purification+of+active+liquid-cultured+Caenorhabditis+elegans+using+a+modified+Baermann+apparatus.">Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus.</a> Parasitology International. 65 (5 Pt B), 580-583 (2016).</li><li>Cornaglia, M., Lehnert, T., Gijs, M. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+systems+for+high-throughput+and+high-content+screening+using+the+nematode+Caenorhabditis+elegans.">Microfluidic systems for high-throughput and high-content screening using the nematode Caenorhabditis elegans.</a> Lab Chip. 17 (22), 3736-3759 (2017).</li><li>Zhu, G., Yin, F., Wang, L., Wei, W., Jiang, L., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+type+2+diabetes-like+hyperglycemia+in+C.+elegans+on+a+microdevice.">Modeling type 2 diabetes-like hyperglycemia in C. elegans on a microdevice.</a> Integrative Biology. 8 (1), 30-38 (2016).</li></ol>

作者没有什么可透露的。

该协议为大型线虫的大规模培养提供了可靠的途径, 获得了定量的结果。文献的结果可以复制, 如代表性结果所示。尽管这个用于收集大型线虫样本的协议似乎是一种直截了当的方法, 但还是有一定的缺陷需要考虑。关于线虫种群的同步问题, 本议定书描述了一种方法, 即用次氯酸钠和氢氧化钠漂白种群, 以破坏线虫, 并完全收获卵子。必须考虑到这种方法可能不适合所有实...

蛋白质分析, 如西方的印迹或质谱, 需要蛋白质的毫克。这种产量需要大规模培养数以百计的线虫, 这可以通过液体培养或固体介质通过洗涤转移线虫。流体和剪切应力诱导上皮钠通道 (ENaC) 的表达, 通过增加钠的吸收增加渗透应力, 可能改变线虫的寿命, 影响代谢分析1.因此, 该协议中的一些关键步骤, 以板块为基础的方法采取减少应力影响实验的变异性考虑。另一方面, 液体培养影响了线虫的表型, 复杂了2线虫的培养和准确数量的收集。此外, 活性物质可以改变的媒介成分, 并可能分布不均匀, 然后到达线虫。关于液体培养的局限性, 本议定书提供了一种新的方法来培养大规模的线虫样本。
线虫是一个模型有机体, 具有独特的网络302神经细胞, 占其所有细胞的 1/33。自引入科学以来, 许多同源和同源基因被描述, 放大它作为医学研究模型的价值。最近, 糖尿病视网膜病变 (前血管损伤) 的神经功能损害的证据已经提出4。线虫缺乏血管, 但包含一个独特的神经网络, 这使得它成为一个合适的模型来调查神经元的变化, 除了血管的。因此, 对于研究糖尿病并发症的一般机制,线虫有特殊的兴趣。糖尿病并发症的生化变化涉及年龄的形成, 这进一步影响了 ROS 的形成, 以应对高血糖5。年龄被发现在C. 线虫和贡献神经损伤6。慢性疾病通常是由复杂的多基因过程引起的, 需要多参数方法来评估其基本机制, 这里举例说明糖尿病并发症的评估。该协议可用于同时获取多个参数, 以及随后使用。通过省略液体与固体培养基之间的形态和代谢差异, 可以提高多参数方法的可比性和重现度。

<table>
	<tbody>
		<tr>
			<td>E. coli OP50</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans N2</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans CL2166</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 60 x 15 mm</td>
			<td>Greiner One</td>
			<td>628161</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric pipet, glas, 10 mL</td>
			<td>Neolab</td>
			<td>E-0413</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>04693124001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-denaturing lysate buffer:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, pH 8</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodiumchloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E5391</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, transparent bottom</td>
			<td>Brand</td>
			<td>781611</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite M200, plate reader</td>
			<td>Tecan</td>
			<td>30017581</td>
			<td></td>
		</tr>
		<tr>
			<td>Zirconium Oxide Beads, 0.5 mm</td>
			<td>Next advance</td>
			<td>ZROB05-RNA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender, homogenizer</td>
			<td>Next advance</td>
			<td>BBX24</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin from porcine gastric mucosa</td>
			<td>Sigma</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymol</td>
			<td>Sigma</td>
			<td>T0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E/ Protease from Streptomyces griseus </td>
			<td>Sigma</td>
			<td>P6911</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P43339</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolidase from Porcine Kidney</td>
			<td>Sigma</td>
			<td>P6675</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminopeptidase from Aeromonas proteolytica</td>
			<td>Sigma</td>
			<td>A8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Merckmillipore</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Materials for plate culture are described in Reference 6.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes

在琼脂盘子上以大规模的方式培养秀丽线虫(线虫) 是费时费力的。该协议描述了一个简单而廉价的方法, 以获得大量的动物, 以分离蛋白质, 进行了西方印迹, 质谱, 或进一步蛋白质组学分析。此外, 在相同的培养条件下, immunostainings 线虫数量的增加和多种分析的整合也很容易实现。另外, 在不同实验条件下的板材之间的转移是方便的。在板块文化中常见的技术是用一根铂丝和用手术刀转移填充的琼脂块来转移单一的线虫.然而, 随着线虫数量的增加, 这些技术变得过于耗时。该协议描述了大规模的C 线虫文化, 包括许多步骤, 以尽量减少样本准备对蠕虫的生理影响。液体和剪切应力可以改变线虫的寿命和代谢过程, 因此需要详细描述关键步骤, 以检索可靠和可重现的结果。线虫是一种模型有机体, 由神经细胞组成, 可达 1/3, 但缺乏血管, 从而提供了单独研究独立于血管控制的神经元改变的可能性。最近, 在血管改变之前发现早期变性糖尿病视网膜病变。因此, 对于研究糖尿病并发症的一般机制,线虫有特殊的兴趣。例如, 在重现性中发现了晚期糖基化终产物 (年龄) 和活性氧 (ROS) 的增加形成. 通过对糖尿病引起的生化变化的研究, 本文介绍了处理足够大小样本以进行更广泛调查的协议。一般而言, 此协议对于需要大的C 线虫数和液体培养不合适的研究是有用的。

这里给出了创建大规模的C. 线虫文化在糖尿病研究中应用的例子。将参数与单个动物进行关联, 而不是将其规范化为总的蛋白质浓度是很有意义的。在需要少量线虫的化验中, 这可以很容易地通过计数线虫来完成。对于一个大规模的C. 线虫文化, 涉及数以百计的线虫每实验组, 这种方法是不方便的。在图 2中, 显示了线虫数量与蛋白?...

Watch this Scientific Journal Video about Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes at JoVE.com

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

本协议描述了一种在固体培养基上大规模培养秀丽线虫的方法。作为液体培养的替代品, 该协议允许在板基培养条件下获得不同尺度的参数。这增加了结果的可比性通过省略液体和固体媒介文化之间的形态学和新陈代谢的区别。

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

plate-based-large-scale-cultivation-caenorhabditis-elegans-sample

Research

JoVE Journal

Biochemistry

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

6.3K 次观看.  Heidelberg University. 本协议描述了一种在固体培养基上大规模培养秀丽线虫的方法。作为液体培养的替代品, 该协议允许在板基培养条件下获得不同尺度的参数。这增加了结果的可比性通过省略液体和固体媒介文化之间的形态学和新陈代谢的区别。

视频: Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

科研

生物化学

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

严格厌氧/缺氧条件下微生物培养的介质制备

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

VMXm 光束线微晶样品制备管道

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

In this protocol, we present an optimized workflow, which combines an efficient and fast sample preparation of many samples. In addition, we provide a step-by-step guide to reduce analytical variations for high-throughput evaluation of metabolic GWAS studies.

大规模多组学全基因组关联研究 （Mo-GWAS）:样品制备和归一化指南

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

摘要

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RNA 结合区质量谱鉴定的样品制备

一种简单的高通量化学筛选方法在秀丽线虫中的应用

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

探头电喷电镀质谱的样品制备

严格厌氧/缺氧条件下微生物培养的介质制备

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

VMXm 光束线微晶样品制备管道

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录

大规模多组学全基因组关联研究（Mo-GWAS）:样品制备和归一化指南

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

摘要

探索更多视频

此视频中的章节

RNA 结合区质量谱鉴定的样品制备

一种简单的高通量化学筛选方法在秀丽线虫中的应用

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

探头电喷电镀质谱的样品制备

严格厌氧/缺氧条件下微生物培养的介质制备

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

VMXm 光束线微晶样品制备管道

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

大规模多组学全基因组关联研究 （Mo-GWAS）:样品制备和归一化指南

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录

大规模多组学全基因组关联研究（Mo-GWAS）:样品制备和归一化指南