Name: polysome profiling in leishmania, human cells and mouse testis
Uploaded: 2018-04-08T15:00:00
Description: Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

作者感谢清李的录音帮助。这项研究得到了来自德州理工大学健康科学中心的启动基金的支持, 以及由翻译神经科学和治疗中心 (CTNT) 授予 PN-CTNT 2017-05 AKHRJDHW A.L.K.;部分由 NIH 授予 R01AI099380 对 K.Z. 詹姆斯 c. 霍夫曼和克里斯汀 r 巴卡是喜赛 (干细胞教育 & 研究的整合中心) 学者并且由节目支持。

所有动物的治疗和组织的处理获得的研究是根据协议的机构动物护理和使用委员会在德州理工大学健康科学中心按照国家研究所健康动物福利准则, 96005 号议定书。请牺牲脊椎动物, 根据动物保育和使用委员会的指导方针准备组织。如果缺乏这样一个委员会, 请参考国家卫生研究院动物福利指南。成人 (> 60 天) C57BL/6 小鼠使用。所有动物和组织都是根据美国德州理工大学卫生科学中心的机构动物护?...

<ol>
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蔗糖梯度结合 RNA 和蛋白质分析 Polysome 分馏是分析个体基因或整个 translatome 的平移状态以及调节平移的蛋白质因子作用的有力方法。机械在正常的生理或疾病状态。Polysomal 分析是一种特别适合于研究生物体中的平移调节的技术, 如 trypanosomatids 包括利什曼原虫, 其中转录控制很大程度上缺乏, 基因表达调控主要发生在翻译过程中。
在这里, 我们描述了一个 polysome 分馏协?...

细胞基因表达调控由转录、转录后水平和翻译后机制控制。在深 RNA 测序方面的进展允许在一个空前的水平上研究基因组范围内的稳态 mRNA 水平。然而, 最近的研究发现, 稳态 mRNA 水平并不总是与蛋白质生产相关1,2。一个单独的成绩单的命运是非常复杂的, 取决于许多因素, 如内部/外部刺激, 压力,等。在蛋白质合成过程中, 基因表达调控为在变化条件下快速反应提供了另一层表达控制。Polysome (或 "polyribosome") 剖面分析, 积极翻译核糖体的分离和可视化, 是研究蛋白质合成调控的有力方法。虽然, 它的第一个实验应用出现在二十世纪六十年代3中, polysome 分析目前是蛋白质翻译研究4中最重要的技术之一。单基因可由多个核糖体转化导致形成 polysome。转录可以停滞在核糖体与放线菌酮5和基因包含不同数量的 polysomes 可以在 polysome 分馏过程中分离的蔗糖梯度离心 6, 7 ,8,9. polysomal 分数的 RNA 分析然后允许测量基因组范围内单个基因的平移状态的变化, 在不同的生理条件下4,7,10. 该方法还用于揭示 5 ' UTR 和 3 ' UTR 序列在 mRNA 可译性的控制中的作用11, 检查 miRNAs 在平移抑制中的角色12, 揭开核糖体合成13 的缺陷, 并了解核糖体相关蛋白与人类疾病的作用14,15。在过去的十年中, 在翻译过程中对基因表达调控的作用越来越大, 这说明了它在人类疾病中的重要性。在癌症、代谢和神经退行性疾病中进行平移控制的证据非常惊人15,16,17,18。例如, eIF4E-dependent 平移控制的失调有助于自闭症相关的赤字15和 FMRP 参与停滞的核糖体基因链接到自闭症14。因此, polysomal 分析是研究多种人类疾病的翻译调节缺陷的重要工具。
不同生理条件下 polysomal 分数的蛋白质分析, 对翻译过程中核糖体相关因素的作用进行了剖析。polysome 分析技术已用于许多物种, 包括酵母, 哺乳动物细胞, 植物和原生动物10,19,20,21。像锥和利什曼原虫这样的原生动物寄生虫表现出有限的基因表达转录控制。他们的基因组被组织成 polycistronic 基因簇, 缺乏启动子调控的转录22。相反, 发展基因表达主要控制在蛋白质的翻译水平和 mRNA 稳定性的 trypanosomatid 物种23,24。因此, 在缺乏转录调控的情况下理解平移控制对这些生物体尤为重要。Polysomal 分析是研究利什曼原虫25、26、27、28中基因表达转录后水平调控的有力工具。
通过实时定量 PCR (RT-qPCR) 和转录的下一代测序以及蛋白质组学技术检测个体基因水平的最新进展, 将 polysomal 分析的解决方案和优势带到了一个新的层次。通过对单个 polysomal 分数的分析, 结合蛋白质组学分析的方法, 可以进一步扩大对各细胞的使用, 以监测整个基因型范围内体细胞的平移状态。这使得在不同的生理和病理条件下, 识别新的分子玩家调节翻译。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型: 寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。本文就不同生物体的细胞裂解物的制备、梯度条件的优化、RNase 抑制剂的选择、qPCR、印迹和 RNA 电泳等方面的应用提出建议, 以分析 polysome 分数。

<table>
	<tbody>
		<tr>
			<td>Instruments:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient master</td>
			<td>Biocomp Instruments Inc.</td>
			<td>108</td>
		</tr>
		<tr>
			<td>Piston Gradient Fractionator</td>
			<td>Biocomp Instruments Inc.</td>
			<td>152</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Gilson, Inc.</td>
			<td>FC203B</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2-FL/Ts2</td>
		</tr>
		<tr>
			<td>2720 Thermal Cycler</td>
			<td>Applied Biosystems by Life Technologies</td>
			<td>4359659</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic Healthcare Co.</td>
			<td>MCO-170A1CUV</td>
		</tr>
		<tr>
			<td>HERATHERM incubator</td>
			<td>Thermo Scientific</td>
			<td>51028063</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet, class II, type A2</td>
			<td>NuAire Inc.</td>
			<td>NU-543-400</td>
		</tr>
		<tr>
			<td>Revco freezer</td>
			<td>Revco Technologies</td>
			<td>ULT1386-5-D35</td>
		</tr>
		<tr>
			<td>Beckman L8-M Ultracentifuge</td>
			<td>Beckman Coulter</td>
			<td>L8M-70</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor SW41</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
		</tr>
		<tr>
			<td>Swing-bucket rotor</td>
			<td>Eppendorf</td>
			<td>A-4-62</td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>F-45-30-11</td>
		</tr>
		<tr>
			<td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4470661</td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>02-671-51B</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triax software&#160;</td>
			<td>Biocomp Instruments Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
		</tr>
		<tr>
			<td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td>
			<td>Seton Scientific</td>
			<td>7030</td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
		</tr>
		<tr>
			<td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td>
			<td>BD</td>
			<td>10020439</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 14 cm</td>
			<td>Thermo Scientific</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 9 cm</td>
			<td>Thermo Scientific</td>
			<td>172931</td>
		</tr>
		<tr>
			<td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td>
			<td>Thermo Scientific</td>
			<td>569-0020</td>
		</tr>
		<tr>
			<td>BioLite 75 cm3 flasks</td>
			<td>Thermo Scientific</td>
			<td>130193</td>
		</tr>
		<tr>
			<td>Nunc 50 mL conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339653</td>
		</tr>
		<tr>
			<td>Chemicals:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol LS</td>
			<td>Ambion by Life Technologies</td>
			<td>10296028</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1378-5KG</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose (DMEM)</td>
			<td>Sigma</td>
			<td>D6429-500ML</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F0926-50ML</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td>
			<td>Acros Organics</td>
			<td>AC413415000</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
		</tr>
		<tr>
			<td>Nonidet P 40 (NP-40)</td>
			<td>Fluka (Sigma-Aldrich)</td>
			<td>74385</td>
		</tr>
		<tr>
			<td>Recombinant Rnasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3993-1MU</td>
		</tr>
		<tr>
			<td>cOmplete Mini EDTA-free protease inhibitors</td>
			<td>Roche Diagnostics</td>
			<td>11836170001</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698-1G</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>194002</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT)</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Fisher Scientific</td>
			<td>BP-1302-10</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>S316-212</td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Life Technologies</td>
			<td>22600050</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma</td>
			<td>P8833-100MG</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-3KG</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma</td>
			<td>T4049-100ML</td>
		</tr>
		<tr>
			<td>Hgh Capacity cDNA Reverse Transcriptase Kit</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4367659</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144SI-212</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26324</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Sigma</td>
			<td>221473-500G</td>
		</tr>
		<tr>
			<td>Anti-RPL11 antibody</td>
			<td>Abcam</td>
			<td>ab79352</td>
		</tr>
		<tr>
			<td>Ribosomal protein S6 (C-8) antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-74459</td>
		</tr>
		<tr>
			<td>1xM199</td>
			<td>Sigma</td>
			<td>M0393-10X1L</td>
		</tr>
		<tr>
			<td>Lithium cloride</td>
			<td>Sigma</td>
			<td>L-9650</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
		</tr>
		<tr>
			<td>Gel Loading Buffer II</td>
			<td>Thermo Scientific</td>
			<td>AM8546G</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Thermo Scientific</td>
			<td>16500-100</td>
		</tr>
		<tr>
			<td>Trichloracetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-100</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS chemiluminescent substrate</td>
			<td>Thermo Scientific</td>
			<td>34580</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>BP531-500</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L5750-1KG</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626-5G</td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A1852-1VL</td>
		</tr>
		<tr>
			<td>Cytosine 5&#39;-triphosphate disodium salt hydrate (CTP)</td>
			<td>Sigma</td>
			<td>C1506-250MG</td>
		</tr>
		<tr>
			<td>Uridine 5&#39;-triphosphate trisodium salt hydrate (UTP)</td>
			<td>Sigma</td>
			<td>U6625-100MG</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma</td>
			<td>G8877-250MG</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>NEB</td>
			<td>M0207S</td>
		</tr>
		<tr>
			<td>Pyrophoshatase</td>
			<td>Sigma</td>
			<td>I1643-500UN</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266-1G</td>
		</tr>
	</tbody>
</table>

polysome profiling in leishmania, human cells and mouse testis

正确的蛋白质表达在适当的时间和正确的数量是正常细胞功能和生存在一个快速变化的环境的基础。长期以来, 基因表达研究以转录水平的研究为主。然而, 基因的稳态水平与蛋白质的生产没有很好的关联, 基因的可译性因条件而变化很大。在某些有机体, 象寄生虫利什曼原虫, 蛋白质表达在翻译水平上主要被调控。最近的研究表明, 蛋白质翻译失调与癌症、新陈代谢、神经变性和其他人类疾病有关。Polysome 分析是研究蛋白质翻译规律的有力方法。它允许测量个体基因的平移状态或在全基因组范围内检查翻译。这种技术的基础是分离 polysomes, 核糖体, 他们的亚基和自由基因在离心的细胞质裂解过程中通过蔗糖梯度。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型-寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。利什曼原虫细胞在悬浮体中自由生长, 培养的人细胞生长在粘附单层, 而小鼠睾丸代表动物组织样本。因此, 该技术适用于所有这些来源。polysomal 分数分析的协议包括通过 rt-pcr 检测个体 mRNA 水平、qPCR 蛋白和用电泳分析核糖体 rna。通过对基因与核糖体在转录水平上的研究, 通过对核糖体相关蛋白的分型质谱分析, 可以进一步扩展该方法。该方法可以很容易地调整到其他生物模型。

在本研究中, 我们描述了 polysomal 分析技术在三种不同来源的应用: 寄生性的利什曼原虫少校、培养的人体细胞和小鼠睾丸。利什曼原虫细胞在悬浮液培养基中自由生长, 培养的人细胞生长在板上的贴膜上, 小鼠睾丸代表组织样本。该方法可以很容易地调整到其他类型的自由生长细胞在悬浮, 不同类型的组织, 或从另一个有机体, 和不同类型的培养细胞。该方法由...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

polysome 分析技术的总体目标是分析个体基因或转录基因在蛋白质合成过程中的平移活动。该方法对健康和多人疾病的蛋白质合成调控、翻译活化和抑制的研究具有重要意义。

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 ...

The overall goal of this technique is to study engagement of indivdual mRNA's with polysomes during translation. Polysome profiling is a powerful technology to study mRNA engagement with ribosomes and polysomes. The technique is important for studies of protein synthesis regulation, translation activation, and repression.Here our team presents the techniques for polysomal fractionation, and analysis of fractions on the example of three organisms. Parasite Leishmania major, cultured human cells, and an animal tissue. This approach consists of four major steps, lysate preparation, sucrose gradient preparation and ultracentrifugation, polysome fractionation, and sample collection.Analysis of the fractions. Cells from different sources are collected, washed and lysed in lysis buffer by passage through a needle or Dounce homogenizer. Centrifugation is used to remove cell debris clarifying the lysate.A continuous sucrose gradient is formed by the mixing of 10%and 50%sucrose solutions in a gradient maker. Lysate is loaded on the top of the gradient. Ultracentrifugation separates mrNA's associated with different number of ribosomes.Which is monitored by a UV Detector during fractionation, forming distinct absorbant spectrums. Lysate preparation. There are some differences in the preparation of the lysate depending on the source.After lysate preparation, procedures are identical regardless of the sample origin. Leishmania major Cytoplasmic Lysate preparation. Place Leishmania major cells in 30 milliliter of medium at the density of 100, 000 cells per milliliter.This step is conducted in a biosafety cabinet. Place the flask with Leishmania culture in the incubator and grow cells at 27 degrees Celsius, until the logarithmic phase. It usually takes about two days to grow.Leishmania major cells are easily visible under the microscope. Add Cycloheximide to the grown Leishmania culture to a final concentration of 100 micrograms per milliliter, to arrest ribosomes on translated mRNAs. Place cells back in the incubator for ten minutes at 27 degrees Celsius.After cycloheximide treatment is completed transfer cells to a 50 milliliter conical tube and spin it 1, 800 g for 8 minutes. Discard supernatant. Wash cells with 30 milliliter of pbs and centrifuge.Discard the supernatant and re-suspend the cells in one milliliter of pbs. Take an aliquot of cells and mix it with the formaldehyde solution. Then count cells by hemocytometer.Transfer the desired number of cells into microfuge tube. Spin the cells at 1, 800 g for 8 minutes at four degrees Celsius. Then discard the supernatant.Re-suspend the cell pellet in one milliliter of lysis buffer containing protease inhibitors and RNAsin. Pass the cells through a 23 gauge needle three times. After passing through the needle, the lysate becomes transparent.Centrifuge at 11, 200 g for ten minutes at four degrees Celsius to clarify lysate. Transfer the clarified lysate to a fresh microfuge tube and keep it on ice until sucrose gradient ultracentrifugation. Cytoplasmic lysate preparation from cultured human cells.Grow HeLa cells in a large plate in the incubator at 37 degrees Celsius, with 5%CO2. Add cycloheximide to grown HeLa cells to the final concentration of 100 micrograms per milliliter to arrest the ribosomes on translated mRNA's. Incubate cells for ten minutes at 37 degrees Celsius with 5%CO2.Aspirate medium and wash plate on ice twice with cold pbs. Add 500 microliters of lysis buffer to the plate and scrape the cells. Transfer the lysed cells to the microfuge tube.Pass the cells through a 23 gauge needle three times. Spin at 11, 200 g for eight minutes to clarify lysate. After centrifugation move the tubes on ice, and transfer supernatant to the new tube.Take an aliquot, dilute it, and measure absorbance at 260 nanometer using a nano drop. If there are multiple samples, adjust the lysates to the same optical density and keep the samples on ice until sucrose gradient ultracentrifugation. Cytoplasmic lysate preparation from mouse testis.Dissect the mouse testis. Make a small incision in the tunica albugenia and collect the seminiforous tubules of the testis and transfer them in the conical tube containing five milliliters of pbs. Mix the tissue vigorously.And allow the tissue to settle on ice for five minutes. Remove the buffer, and wash it with pbs two more times. Transfer the seminiferous tubules in a two milliliter tube and spin them down at 500 g for one minute.Remove the supernatant. Add 500 microliter lysis buffer to the tubules and use a pipet to disrupt the tissue. Transfer the suspension to a small volume dounce homogenizer.Disrupt the tissue with seven to eight strokes of the glass pestle. Transfer the lysate to a micro centrifuge tube. Centrifuge the sample at 12, 000 g at four degrees Celsius for eight minutes to clear the lysate.Transfer the supernatant into a new tube, and keep it on ice. Then, proceed with sucrose gradient preparation. Sucrose gradient preparation.Place an ultracentrifuge tube into the microblock and draw the line along the upper level of the block. Then transfer the tube into a stable rack. Take a ten milliliter syringe, with the layering device attached and fill the syringe with 10%sucrose solution.Gently release it at the bottom of the ultracentrifuge tube until sucrose solution reaches the mark. Fill another syringe with 50%sucrose solution and carefully insert its layering device through the 10%sucrose layer to the bottom of the tube. Gently release sucrose solution, starting from the bottom until it reaches the mark on the tube.Then seal the tube with provided cap. To prepare sucrose gradient, turn the gradient maker device on. Then level the plate using the up or down buttons and press done.After leveling the plate press grad. Go to the list of gradient menu and select the SW41 rotor. Then choose desired sucrose gradient.Press use. Place magnet base tube holder on the gradient maker. Then transfer the tube into the holder.Up to six gradients can be prepared at the same time. Press run. The gradient maker rotates the tubes at the program speed and angles, forming a linear gradient.It will take only a few minutes to prepare the gradient. When the process is completed, place the tube in a rack. Remove the volume, equal of sample volume from the top of the ultracentrifuge tubes with sucrose gradients.Carefully load 500 microliters of the lysate on top. Place the tubes in rotor buckets and balance them. Centrifuge at 260, 000 g for two hours at four degrees Celsius.Polysome fractionation and sample collection. After ultracentrifugation, place the tubes in the rotor buckets on ice. Turn the fraction collector and the fractionator on.Click scan on the fractionator menu. Put a rack with collection tubes into fraction collector. Fill up a rinse reservoir, on the side of the fractionator with deionized water.Press rinse key for ten seconds to rinse the pump. Attach a rinse adapter with the syringe filled with water to the piston for the calibration. Open the fractionator software on the computer.Press calibrate. Use the default setting and press Ok.Be ready to inject water from the syringe. Press Ok to do calibration.Immediately start injecting water for the next five seconds. The sign Zero calibration completed will appear. Remove a rinse adapter with the syringe and attach a tip to the piston of the fractionator.Open the brass air valve and press air key for ten seconds to dry air tubing and flow cell. Close the air valve. Take the gradient tube from the rotor bucket and place it in the rack.Apply the tube holder cap to the top of the tube, and carefully move the tube into the tube holder and lock it. Place the holder under the piston. Often, polysomal bands can be seen by eye.Introduce the desired settings for fraction numbers and volume. Name the file and press Ok, then go to graph button. In the next window press start scan.Settings will appear, and press Ok.The collector will move from the gutter to the first fraction, and the piston will move into the tube. When the piston will reach the top of the gradient it will slow to your selected speed, and fractions will be collected. Usually, we collect 24 fractions at 500 microliters each.When completed, the piston will move out of the gradient tube. Open the brass air valve. Press air key on the fractionator to retrieve the last fraction.At the end of the run you will see the absorbance profile of the gradient. Analysis of the fractions. Fractions obtained can be used for RNA and Protein analysis.RNA can be analyzed by electrophoresis followed by Northern Blot or used for cDNA production, followed by RT-qPCR reaction to analyze association of individual mRNA's with polysomes. Next generation sequencing can be used to analyze the translational status of mRNA's on a transcriptome scale. For protein analysis of polysomal fractions, proteins are precipitated with tricluricedic acid to concentrate them.Then proteins are analyzed by Western Blotting, or by Mass Spec at the proteome level.Results. Here we present the results of polysome fractionation and analysis on the examples of three organisms. Leishmania, cultured human cells, and mouse tissue.Polysome fractionation was used to study Sherp and Tubulin mRNA's association with ribosomes during translation and Leishmania major. The absorbance graph for the fractionation has a distinct shape with typical peaks for ribosome sub-units 40S and 60S, monosomes 80S, and polysomes. Aliquots of the fractions were combined into three groups.Pre-polysomes, light polysomes, and heavy polysomes. The mRNA relative levels were studied by a real time quantitative PCR. The data suggests that the Tubulin mRNA is actively translated.As it was mostly associated with heavy and light polysomes. While the translation of Sherp mRNA is less active. It was mostly found in association with pre-polysomes and light polysomes.Thus, this experiment clearly demonstrates the ability of the technique to study an individual mRNA engagement in translation. This figure presents a typical absorbance graph of the HeLa cells polysome fractionation. The proteins were concentrated by precipitation with 10%trichloroacetic acid.The small sub unit ribosomal protein RPS6, and large sub unit ribosomal protein RPL11 were detected by Western Blot. Their distribution correlated well with the distinct peaks, on the absorbance spectrum. On this figure, we present an example of the detection of ribosomal RNAs by electrophoresis in fractionated polysomes form the mouse testis.Conclusions.Polysomal profiling is a versatile technique, that can be used to analyze translational state of individual mRNAs, examine ribosome-associated proteins, and study translational regulation in different model organisms under different conditions.

Research

JoVE Journal

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

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

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 ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Ganglioside Extraction, Purification and Profiling

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Polysome Profiling without Gradient Makers or 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 ...