Name: horizontal gel electrophoresis for enhanced detection of protein-rna complexes
Uploaded: 2017-07-28T14:00:00
Description: Watch this Scientific Journal Video about Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes at JoVE.com

我们感谢Laura Vanderploeg准备数字。 NSF grant 1050395和NIH grant（R21HD076828）支持在Sheets实验室工作。 NIH授权R01GM117237和R01GM117008支持莱德实验室的工作。 Megan Dowdle通过美国威斯康星大学麦迪逊分校研究生院和生物技术培训项目通过国立卫生研究院国家综合医学研究所（T32GM008349）得到了SciMed GRS高级机会奖学金的支持。

水平天然聚丙烯酰胺凝胶的制备4 。 <ol><li> 准备所需材料 <ol><li>对于水平凝胶装置，使用具有27cm×21cm托盘的凝胶盒（37cm×24cm），并容纳两个24孔梳子。此设置提供了总共48个样本，可以同时进行分析。 </li><li>准备以下试剂：40％丙烯酰胺 - 双19：1,5x TBE（Tris-Borate-EDTA pH 8.0），TEMED和10％APS（过硫酸铵）。 </li></ol></li><li> 生成10％天然丙烯酰胺凝胶。 <ol><l...

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	<li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Dahlberg, A. E., Dingman, C. W., Peacock, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+characterization+of+bacterial+polyribosomes+in+agarose-acrylamide+composite+gels.">Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels.</a> J Mol Biol. 41 (1), 139-147 (1969).</li><li>Hellman, L. M., Fried, M. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Characterization+of+Binding+Oxytricha+nova+Single+Strand+Telomere+DNA+with+the+Alpha+Protein+N-terminal+Domain.">Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain.</a> J Mol Biol. 359 (5), 1217-1234 (2006).</li><li>Zhang, Y., Park, S., Blaser, S., Sheets, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+RNA+binding+and+translational+repression+by+the+Bicaudal-C+regulatory+protein.">Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.</a> J Biol Chem. 289 (11), 7497-7504 (2014).</li><li>Gamberi, C., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bic-C+family+of+developmental+translational+regulators.">The Bic-C family of developmental translational regulators.</a> Comp Funct Genomics. , 141386 (2012).</li><li>Saffman, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+translation+of+oskar+in+oocytes+lacking+the+RNA-binding+protein+bicaudal-C.">Premature translation of oskar in oocytes lacking the RNA-binding protein bicaudal-C.</a> Mol Cell Biol. 18 (8), 4855-4862 (1998).</li><li>Chicoine, J., 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=Bicaudal-C+recruits+CCR4-NOT+deadenylase+to+target+mRNAs+and+regulates+oogenesis,+cytoskeletal+organization,+and+its+own+expression.">Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression.</a> Dev Cell. 13 (5), 691-704 (2007).</li><li>Zhang, 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=Bicaudal-C+spatially+controls+translation+of+vertebrate+maternal+mRNAs.">Bicaudal-C spatially controls translation of vertebrate maternal mRNAs.</a> RNA. 19 (11), 1575-1582 (2013).</li><li>Maisonneuve, C., 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=Bicaudal+C,+a+novel+regulator+of+Dvl+signaling+abutting+RNA-processing+bodies,+controls+cilia+orientation+and+leftward+flow.">Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow.</a> Development. 136 (17), 3019-3030 (2009).</li><li>Tran, U., 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+RNA-binding+protein+bicaudal+C+regulates+polycystin+2+in+the+kidney+by+antagonizing+miR-17+activity.">The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.</a> Development. 137 (7), 1107-1116 (2010).</li><li>Pagano, J. 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非变性聚丙烯酰胺凝胶是用于研究蛋白质-RNA相互作用和传统上这些凝胶是垂直电泳2,3的宝贵工具。我们已经使用该替代创建和水平1，4，6，7，15电泳天然聚丙烯酰胺凝胶的协议的修改。与荧光标记的RNA底物组合使用的这些变化为?...

电泳迁移率变动分析（EMSAs）已被证明是一种宝贵的生化工具分析特定的蛋白质-核酸相互作用1，2，3。这些检测可以提供关于蛋白质与RNA或DNA的结合亲和力的重要信息3 ，核酸 - 蛋白质复合物1的组分化学计量学，并通过底物竞争实验提供关于RNA结合蛋白的结合特异性的重要新见解1 。 用于这些测定的传统实验装置包括将纯化的蛋白质与放射性标记的RNA底物混合。然后用不变性（天然的）聚丙烯酰胺凝胶分析所得的复合物，将其倒入两个玻璃板之间，然后在垂直装置3中进行样品电泳。尽管这种方法已被彻底地用于提供蛋白质与核酸结合的基础的生物化学机制的重要见解，但它也具有一些限制。例如，这种基本策略具有相对低的吞吐量，并且不容易适用于需要并行分析许多结合反应的应用。此外，与传统的立式设备是有挑战性的电泳3,4期间潜在地监测在多个时间络合物。 这里，我们提出了使用铸造成平板装置天然聚丙烯酰胺凝胶，水平电泳和荧光标记的RNA底物4，5，6，7的EMSA分析的适应。将这些相对简单的修改结合到基本的strategy提供了一些强大的优势。特别地，水平平板格式很容易同时分析几十个样本4 。此外，对于一些RNA-蛋白复合物，例如在Bicaudal-C蛋白和其RNA底物之间形成的那些在水平凝胶中电泳的那些，提供了分辨不同RNA-蛋白复合物的增加的能力，并将它们与未结合的RNA底物区分开来。

<table border="0" cellpadding="0" cellspacing="0" width="1432">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Horizontal Gel Box</td>
			<td style="width:112px;">OWL</td>
			<td style="width:116px;">N/A</td>
			<td style="width:831px;">Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel boxes. Catalog Number: A2-BP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well large large horizontal gel electrophoresis combs</td>
			<td>OWL</td>
			<td>N/A</td>
			<td>Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel box combs. Catalog Number: A2-24C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerpac 300</td>
			<td>Bio-Rad</td>
			<td>1655050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Protean II Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>165-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InstaPAGE-19 40% 19:1 Acrylamide/Bis</td>
			<td>IBI</td>
			<td>IB70015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>IBI</td>
			<td>IB70120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APS</td>
			<td>IBI</td>
			<td>IB70080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNAs</td>
			<td>Ambion</td>
			<td>AM7119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescein labeled RNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Order can be made custom to length and desired sequence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA tetrasodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>E5391-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base Ultrapure</td>
			<td>US Biological</td>
			<td>T8600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>BP168-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC</td>
			<td>Sigma-Aldrich</td>
			<td>1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>3483 12 3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

horizontal gel electrophoresis for enhanced detection of protein-rna complexes

天然聚丙烯酰胺凝胶电泳是分子生物学的基础工具，广泛应用于RNA-蛋白质相互作用的生物化学分析。这些相互作用传统上用两片玻璃板和垂直电泳样品之间产生的聚丙烯酰胺凝胶进行分析。然而，聚丙烯酰胺凝胶在托盘中浇铸并水平电泳提供了几个优点。例如，用于分析荧光RNA底物和特定蛋白质之间的复合物的水平凝胶可以在电泳进行时多次成像。这提供了在实验期间在几个点监测RNA-蛋白复合物的独特机会。此外，水平凝胶电泳可以并行分析许多样品。这可以大大促进时间实验以及同时分析多个反应以比较不同的组件和条件。我们这里制定详细的方案，用于生成和使用水平天然凝胶电泳分析RNA-蛋白质相互作用。

为了证明水平天然凝胶电泳的功能和多功能性，我们分析了非洲爪蟾 Bicaudal-C（Bicc1）蛋白与含有Bicc1结合位点的荧光标记RNA的结合。 Bicc1蛋白用作特异性mRNA翻译阻遏期间动物发展9，10，11，12的母体阶段以及特定器官的成人13,14</su...

Watch this Scientific Journal Video about Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes at JoVE.com

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

天然聚丙烯酰胺凝胶电泳是分析RNA蛋白相互作用的基础工具。传统上大多数实验都使用垂直凝胶。然而，水平凝胶提供了几个优点，例如在电泳期间监测复合物的机会。我们提供生成和使用水平天然凝胶电泳的详细协议。

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

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Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

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Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

使用非变性聚丙烯酰胺凝胶电泳人的MxA多亚基蛋白复合物的表征

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

蛋白质tRNA琼脂糖凝胶延迟测定分析<em&gt;ñ</em<sup&gt; 6</sup&gt; - 磺酰氨基甲酰腺苷TcdA功能

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

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Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...