Name: polysome profiling without gradient makers or fractionation systems
Uploaded: 2021-06-01T14:00:00
Description: Watch this Scientific Journal Video about Polysome Profiling without Gradient Makers or Fractionation Systems at JoVE.com

作者感谢Percy Tumbale博士和Melissa Wells博士对这份手稿的批判性阅读。这项工作得到了美国国家卫生研究院校内研究计划的支持;美国国家环境健康科学研究所（NIEHS;ZIA ES103247 至 R.E.S）。

1. 制备7%-47%蔗糖梯度
注意：蔗糖梯度的线性范围可以根据所使用的细胞类型进行修改，以实现更好的分离。该协议针对 酿酒酵母的多体谱进行了优化。
<ol><li>在蔗糖梯度缓冲液（20 mM Tris-HCl pH 7.4、60 mM KCl、10 mM MgCl2 和 1 mM DTT）中制备 7% 和 47% 蔗糖储备溶液。过滤器通过0.22μm过滤器对蔗糖储备溶液进行灭菌，并储存在4°C。</li>
	<li>通过按 表1中所?...

<ol>
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S., McCormick, C., Cheng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+profiling+analysis+of+mRNA+and+associated+proteins+engaged+in+translation.">Polysome profiling analysis of mRNA and associated proteins engaged in translation.</a> Current Protocols in Molecular Biology. 125 (1), 79 (2019).</li><li>Liang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome-profiling+in+small+tissue+samples.">Polysome-profiling in small tissue samples.</a> Nucleic Acids Research. 46 (1), 3 (2018).</li><li>Karamysheva, Z. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+profiling+in+leishmania,+human+cells+and+mouse+testis.">Polysome profiling in leishmania, human cells and mouse testis.</a> Journal of Visualized Experiments: JoVE. , e57600 (2018).</li><li>Jin, H. Y., Xiao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Integrated+polysome+profiling+and+ribosome+profiling+method+to+investigate+in+vivo+translatome.">An Integrated polysome profiling and ribosome profiling method to investigate in vivo translatome.</a> Methods in Molecular Biology. 1712, 1-18 (2018).</li><li>Chasse, H., Boulben, S., Costache, V., Cormier, P., Morales, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+translation+using+polysome+profiling.">Analysis of translation using polysome profiling.</a> Nucleic Acids Research. 45 (3), 15 (2017).</li><li>Aboulhouda, S., Di Santo, R., Therizols, G., Weinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate,+streamlined+analysis+of+mrna+translation+by+sucrose+gradient+fractionation.">Accurate, streamlined analysis of mrna translation by sucrose gradient fractionation.</a> Bio-protocol. 7 (19), 2573 (2017).</li><li>Kudla, M., Karginov, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mRNA+translation+by+polysome+profiling.">Measuring mRNA translation by polysome profiling.</a> Methods in Molecular Biology. 1421, 127-135 (2016).</li><li>Faye, M. D., Graber, T. E., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+selective+mRNA+translation+in+mammalian+cells+by+polysome+profiling.">Assessment of selective mRNA translation in mammalian cells by polysome profiling.</a> Journal of Visualized Experiments: JoVE. (92), e52295 (2014).</li><li>Coudert, L., Adjibade, P., 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=Analysis+of+translation+initiation+during+stress+conditions+by+polysome+profiling.">Analysis of translation initiation during stress conditions by polysome profiling.</a> Journal of Visualized Experiments: JoVE. (87), e51164 (2014).</li><li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+fractionation+to+analyze+mRNA+distribution+profiles.">Polysome fractionation to analyze mRNA distribution profiles.</a> Bio-protocol. 7 (3), 2126 (2017).</li><li>Chikashige, 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=Gcn2+eIF2alpha+kinase+mediates+combinatorial+translational+regulation+through+nucleotide+motifs+and+uORFs+in+target+mRNAs.">Gcn2 eIF2alpha kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in target mRNAs.</a> Nucleic Acids Research. 48 (16), 8977-8992 (2020).</li><li>Ingolia, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+footprint+profiling+of+translation+throughout+the+genome.">Ribosome footprint profiling of translation throughout the genome.</a> Cell. 165 (1), 22-33 (2016).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Molecular and Cellular Biology. 13 (12), 7901-7912 (1993).</li><li>Lo, Y. H., 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=Cryo-EM+structure+of+the+essential+ribosome+assembly+AAA-ATPase+Rix7.">Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7.</a> Nature Communications. 10 (1), 513 (2019).</li><li>Pillon, M. C., Sobhany, M., Borgnia, M. J., Williams, J. G., Stanley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grc3+programs+the+essential+endoribonuclease+Las1+for+specific+RNA+cleavage.">Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (28), 5530-5538 (2017).</li><li>Woolford, J. L., Baserga, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+biogenesis+in+the+yeast+Saccharomyces+cerevisiae.">Ribosome biogenesis in the yeast Saccharomyces cerevisiae.</a> Genetics. 195 (3), 643-681 (2013).</li><li>Klinge, S., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+assembly+coming+into+focus.">Ribosome assembly coming into focus.</a> Nature Reviews. Molecular Cell Biology. 20 (2), 116-131 (2018).</li><li>Bassler, J., Hurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+ribosome+assembly.">Eukaryotic ribosome assembly.</a> Annual Review of Biochemistry. 88, 281-306 (2019).</li><li>Kressler, D., Hurt, E., Bassler, 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+puzzle+of+life:+Crafting+ribosomal+subunits.">A puzzle of life: Crafting ribosomal subunits.</a> Trends in Biochemical Sciences. 42 (8), 640-654 (2017).</li><li>Prattes, M., Lo, Y. H., Bergler, H., Stanley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+nascent+ribosome:+AAA-ATPases+in+eukaryotic+ribosome+biogenesis.">Shaping the nascent ribosome: AAA-ATPases in eukaryotic ribosome biogenesis.</a> Biomolecules. 9 (11), 715 (2019).</li><li>Pillon, M. C., Sobhany, M., Stanley, R. E. <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+the+molecular+crosstalk+within+the+essential+Grc3/Las1+pre-rRNA+processing+complex.">Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex.</a> RNA. 24 (5), 721-738 (2018).</li><li>Hu, W., Coller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+for+determining+mRNA+and+ribosome+association+in+Saccharomyces+cerevisiae.">Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae.</a> Methods in Enzymology. 530, 193-206 (2013).</li><li>He, S. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+of+mammalian+cells.">Polysome analysis of mammalian cells.</a> Methods in Enzymolog. 530, 183-192 (2013).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> The Journal of Biological Chemistry. 278 (9), 6985-6991 (2003).</li><li>Serikawa, K. A., 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=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Molecular and Cell Proteomics: MCP. 2 (3), 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., 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=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li></ol>

这里描述了一种无需使用昂贵的自动分馏系统即可创建多聚体谱的方法。这种方法的优点是，它使没有自动分级分离系统的实验室可以使用多聚体分析。与专用密度分级分离系统相比，该方案的主要缺点是繁琐的手工分馏和灵敏度降低。
该方案需要仔细制备蔗糖梯度，其分辨率足以分离核糖体亚基，单体和多聚体。在制备蔗糖梯度时，在加载梯度层时不要引入气泡至关重要?...

核糖体是巨型道尔顿核糖核蛋白复合物，执行将mRNA翻译成蛋白质的基本过程。核糖体负责进行细胞内所有蛋白质的合成。真核核糖体包括两个亚基，根据其沉降系数指定为小核糖体亚基（40S）和大核糖体亚基（60S）。完全组装的核糖体被指定为80S单体。多聚体是参与翻译单个mRNA分子的核糖体组。通过蔗糖密度梯度离心分馏多聚体是一种强大的方法，用于创建核糖体谱，鉴定与翻译核糖体相关的特定mRNA，并分析多聚体相关因子1，2，3，4，5，6，7，8，9，10，11，12，13.该技术通常用于从单个核糖体、核糖体亚基和信使核糖核蛋白颗粒中分离多聚体。从分级分离中获得的图谱可以提供有关多聚体14的翻译活性和核糖体15，16，17的组装状态的宝贵信息。
核糖体组装是一个非常复杂的过程，由一组称为核糖体组装因子18，19，20，21的蛋白质促进。这些因子通过与许多其他蛋白质（包括ATP酶，内切和外切核酸酶，GTP酶，RNA解旋酶和RNA结合蛋白）的相互作用，在核糖体生物发生过程中执行广泛的功能22。多聚体分级分离一直是用于研究这些因素在核糖体组装中的作用的有力工具。例如，该方法已被用于证明多核苷酸激酶Grc3（一种前rRNA加工因子）的突变如何对核糖体组装过程产生负面影响17，23。多聚体分析还强调并展示了ATPase Rix7中的保守基序对核糖体生产至关重要16。
多聚体分级分离的过程始于从感兴趣的细胞中制备可溶性细胞裂解物。裂解物含有RNA、核糖体亚基和多聚体，以及其他可溶性细胞成分。在超速离心管内进行连续的线性蔗糖梯度。将细胞裂解物的可溶性部分轻轻上样到蔗糖梯度管的顶部。然后对装载的梯度管进行离心，离心通过重力在蔗糖梯度内按大小分离细胞成分。较大的分量比较小的分量更深入梯度。梯度的顶部容纳较小，移动较慢的细胞成分，而较大，快速移动的细胞成分位于底部。离心后，将管中的内容物收集为馏分。该方法可有效分离核糖体亚基、单体和多聚体。然后通过测量254nm波长处的光谱吸光度来确定每个级分的光密度。绘制吸光度与分数数的关系图可生成多聚体谱图。
可以使用梯度制作器生成线性蔗糖密度梯度。离心后，通常对梯度进行分馏，并使用自动密度分馏系统3，7，13，24，25测量吸光度。虽然这些系统在生产多聚体谱方面效果很好，但它们价格昂贵，并且对一些实验室来说成本过高。这里提出了一种在不使用这些仪器的情况下生成多聚体谱的协议。相反，该协议利用大多数分子生物学实验室中通常可用的设备。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic Fractionator</td>
			<td>Brandel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clariostar Multimode Plate Reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Invitrogen</td>
			<td>15508-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beads, acid washed</td>
			<td>Sigma Aldrich</td>
			<td>G8772</td>
			<td>425&#8211;600 &#956;m</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride, 1 M</td>
			<td>KD Medical</td>
			<td>CAC-5290</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 22 G, Metal Hub</td>
			<td>Hamilton Company</td>
			<td>7748-08</td>
			<td>custom length 9 inches, point style 3</td>
		</tr>
		<tr>
			<td>Optima XL-100K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Test Tube Peg Rack</td>
			<td>Fisher Scientific</td>
			<td>14-810-54A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q33228</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32855</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>N8080119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SW41 Swinging Bucket Rotor Pkg</td>
			<td>Beckman Coulter</td>
			<td>331336</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 3 mL</td>
			<td>Coviden</td>
			<td>888151394</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris, 1 M,&#160; pH 7.4</td>
			<td>KD Medical</td>
			<td>RGF-3340</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Star Microplate, 96 wells</td>
			<td>Greiner Bio-One</td>
			<td>655801</td>
			<td></td>
		</tr>
	</tbody>
</table>

polysome profiling without gradient makers or fractionation systems

通过蔗糖密度梯度离心进行多聚体分级分离是一种强大的工具，可用于创建核糖体谱，鉴定核糖体翻译的特定mRNA，并分析多聚体相关因子。虽然自动梯度测定器和梯度分馏系统通常与这种技术一起使用，但这些系统通常价格昂贵，并且对于资源有限或由于不经常或偶尔需要执行此方法而无法证明费用合理的实验室来说成本过高。在这里，提出了一种协议，使用大多数分子生物学实验室中可用的标准设备（无需专门的分级分离仪器）可重复地生成多聚体谱。此外，还提供了使用和不使用梯度分馏系统生成的多聚体谱图的比较。讨论了优化和产生可重复的多体谱的策略。 酿酒酵母 在本协议中用作模式生物。然而，该协议可以很容易地修改和调整，以产生许多不同的生物体和细胞类型的核糖体谱。

图3显示了三个代表性的多聚体谱。所有配置文件都来自相同的酵母菌株。典型的多体谱将具有40S，60S和80S核糖体亚基以及多聚体的良好分离峰。每个核糖体亚基和多聚体峰的嵴在每个轮廓上都很明显（图3）。自动密度分馏系统的代表性曲线如图3A所示。用于生成该轮廓的蔗糖梯度是按照本协议中所述手动制备的。...

Watch this Scientific Journal Video about Polysome Profiling without Gradient Makers or Fractionation Systems at JoVE.com

Polysome Profiling without Gradient Makers or Fractionation Systems

该协议描述了如何在不使用自动梯度制作器或梯度分级分离系统的情况下生成多聚体谱。

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 ...

该协议描述了如何生成多聚体图谱，该图谱揭示了有关细胞内核糖体活性的分子细节。该技术允许用户获得多聚体谱提供的有价值的信息，而多聚体谱无法访问自动梯度分级分离系统。在准备渐变时花点时间，并将渐变存储在一个位置，当它们进入线性渐变时，它们不会被压缩机或其他机械操作破坏。首先，在蔗糖梯度缓冲液中制备7%和47%蔗糖的储备溶液。过滤器通过0.22微米过滤器对蔗糖储备溶液进行灭菌。通过分配并混合 7% 和 47% 的储备溶液，制备 14 毫升 17、27% 和 37% 蔗糖溶液。将六个聚丙烯离心管放入全视图试管架中。确保管子之间有足够的空间，以便一个管子的动作不会干扰其他管子。将带有钝头的 9 英寸、22 号针头连接到 3 毫升注射器上，并进行测试填充和分配，以确保注射器可以在设置梯度之前容纳蔗糖溶液而不会滴落。在每个离心管的底部加入两毫升7%蔗糖。然后，通过将针尖定位在管底附近，在 7% 溶液下方加入两毫升 17% 蔗糖。小心而缓慢地分配溶液。用27%，37%和47%蔗糖溶液各两毫升重复该过程，确保每层都可以通过标记密度分离的线与另一层区分开来。将梯度在四摄氏度下储存过夜，使它们沉淀成连续的、增加百分比的蔗糖。在酵母，酿酒酵母细胞的生长和收获之后，将细胞重悬于冷冻的700微升多聚体提取缓冲液中。加入100单位的RNA酶抑制剂。然后，加入 400 微升预冷玻璃珠，大小约为 425 至 600 微米。将混合物转移到装有玻璃珠的1.5毫升离心管中，并通过在打珠器中剧烈搅拌五分钟来破坏酵母细胞。破碎细胞后，通过离心澄清裂解物。使用基于荧光的RNA检测系统，通过测量260纳米处的吸光度来确定澄清裂解物中RNA的浓度。确保RNA浓度为每微升0.5至1微克。如果RNA浓度太低，请减少用于重悬细胞的提取缓冲液的体积。小心地将裂解物加载到梯度的顶部。将移液器吸头靠在聚丙烯管顶部的内壁上。倾斜试管，将裂解物缓慢分配到梯度的顶部，滴在壁上。轻轻地将试管放入水平吊篮转子的预冷桶中，然后离心梯度。离心后，小心地从摆动桶转子上取下离心管，并将它们放入管架中。标记96孔板以储存馏分并在冰上预冷。从梯度顶部开始收集 100 或 200 微升馏分，小心地将移液器吸头插入梯度顶部，并收集馏分直到整个梯度被等分。用分光光度计测量每个级分在254纳米处的吸光度，将7%和47%蔗糖溶液作为空白。通过绘制分数数与吸光度的关系来创建多聚体谱图。显示了来自酿酒酵母的三种代表性多体谱。每个核糖体亚基和多聚体峰的嵴在每个轮廓上都很明显。此处显示了自动密度分馏系统的代表性轮廓。该曲线由连续的吸光度曲线产生，因为蔗糖梯度被追逐溶液自下而上，通过检测器流通池并分馏收集。此处显示了手动分馏 200 和 100 μL 样品生成的多聚体谱图。在此过程中要记住的最重要的事情是不要破坏梯度。注意不要通过过快分配溶液来引入气泡或破坏梯度。按照此过程，可以从梯度中提取RNA，以进行进一步分析，以确定与活性核糖体相关的mRNA

Research

JoVE Journal

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

科研

生物化学

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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 ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

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 ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

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 ...