Name: horizontal gel electrophoresis for enhanced detection of protein-rna complexes
Uploaded: 2017-07-28T14:00:00.000+00:00
Duration: 6 min 36 s
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><li>在500mL烧瓶中，加入80mL 5x TBE，100mL 40％丙烯酰胺和水，至最终体积为400mL。 </li><li>加入4 mL 10％APS和400μLTEMED。混合好</li><li>将混合物倒入水平铸造托盘中，使凝胶聚合30分钟。凝胶将约2厘米厚。 注意:凝胶可以放入通风橱中加速聚合。聚合后，薄层的液体将保留在凝胶的顶部。 </li><li>将液体倒出并用运行缓冲液冲洗。小心地取出梳子，使用注射器用运行缓冲液冲洗孔。 </li></ol></li><li> 准备运行缓冲区。 <ol><li>为凝胶盒准备2 L 1x TBE运行缓冲液。用1600 mL稀释400 mL 5XTBE并混合。将1x TBE缓冲液放在冷藏室中冷却。 </li></ol></li><li> 预运行凝胶 <ol><li>将2升冷运行缓冲液加入凝胶盒中并插入凝胶。 </li><li>在冷藏室中，在4℃下将凝胶在120V预处理1小时。在此期间，制备结合反应。 </li></ol></li></ol>绑定反应8 <ol><li> 2.1。制备以下反应组分。 20 mM DTT 10mM BSA 10mM酵母tRNA 5X结合缓冲液（50mM HEPES pH 8.0,5mM EDTA，0mM KCl，0.2％Tween-20） 购自商业上购买的100nM荧光素标记的RNA 浓缩Bicaudal-C蛋白 在DEPC处理的H 2 O中的50％甘油 DEPC处理的H 2 O </li><li> 在无RNA酶的微量离心管中装配结合反应组分。 <ol><li>向管中加入5μL的10mM BSA，5μL的20mM DTT，5μL的10mM酵母tRNA，10μL的5x结合缓冲液。 </li></ol></li><li>在50μL中加入最终浓度为250 nM的蛋白。加水至最终体积为45μL。 </li><li>加入5μL荧光标记的RNA底物。使用商业上购买的3&#39;荧光素标记的32核苷酸底物。 </li><li>在室温下混合并在黑暗中孵育30分钟。 </li></ol> 3.天然水平聚丙烯酰胺凝胶样品分析<ol><li>在每次反应中，加入15μL50％甘油，轻轻混匀。 </li><li> Carefu每次反应加入30μL凝胶。可以装入单个孔的样品量根据凝胶的厚度和孔的大小而变化。我们已经将体积少至15μL和多达45μL加入到用24孔梳子制成的单个孔中，并且倾倒到2cm的厚度。 </li><li> 将电源连接到凝胶装置开关电源并调节电压。在120℃下在4℃下运行凝胶。 注意:对于水平凝胶装置，其结果为约5V / cm。对于垂直凝胶装置，这是16 V / cm。 <ol><li>为了防止荧光标记的RNA的破坏或漂白，在黑暗中进行电泳。 </li><li>根据正在分析的RNA-蛋白质复合物的特异性而改变电泳时间。 注意:水平EMSA分析的主要优点是可以停止电泳，凝胶成像，然后再进行电泳l可以放回装置，电泳可以持续更长时间。 </li></ol></li></ol>凝胶成像<ol><li>使用任何设计用于检测和成像荧光底物的仪器进行分析。 </li><li>将凝胶放在成像仪上。 </li><li>调整成像器设置。对于荧光素标记的RNA配体，请使用以下设置:FAM（波长473 nm），750倍光电倍增管电压（PMT），100μm像素大小。 </li><li>扫描凝胶以捕获图像。 </li></ol>

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作者没有什么可以披露的。

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><tbody><tr><td>Horizontal Gel Box</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 boxes. Catalog Number: A2-BP</td></tr><tr><td>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><td>Powerpac 300</td><td>Bio-Rad</td><td>1655050</td><td></td></tr><tr><td>Mini-Protean II Electrophoresis Cell</td><td>Bio-Rad</td><td>165-2940</td><td></td></tr><tr><td>InstaPAGE-19 40% 19:1 Acrylamide/Bis</td><td>IBI</td><td>IB70015</td><td></td></tr><tr><td>TEMED</td><td>IBI</td><td>IB70120</td><td></td></tr><tr><td>APS</td><td>IBI</td><td>IB70080</td><td></td></tr><tr><td>Yeast tRNAs</td><td>Ambion</td><td>AM7119</td><td></td></tr><tr><td>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><td>EDTA tetrasodium salt hydrate</td><td>Sigma-Aldrich</td><td>E5391-1KG</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Tris Base Ultrapure</td><td>US Biological</td><td>T8600</td><td></td></tr><tr><td>Boric Acid</td><td>Fisher Scientific</td><td>BP168-500</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>BP366-1</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>DEPC</td><td>Sigma-Aldrich</td><td>1609-47-8</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>3483 12 3</td><td></td></tr><tr><td>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 ...

horizontal-gel-electrophoresis-for-enhanced-detection-protein-rna

Research

JoVE Journal

Biochemistry

11.9K 次观看.  University of Wisconsin School of Medicine and Public Health. 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。 

视频: Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

生物化学

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

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 tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

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

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

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 ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

生物学

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究:IRE / IRP示例

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, underlining the importance of post-transcriptional regulation of gene expression orchestrated by RBPs. To elucidate mechanistic roles of an RBP, crosslinking immunoprecipitation (CLIP) methodology can be used to capture its endogenous direct RNA targets and define the actual interaction sites. The enhanced CLIP (eCLIP) is a newly-developed method that offers several advantages over the conventional CLIPs. However, the use of eCLIP has so far been limited to cell lines, calling for expanded applications. Here, we employed eCLIP to study MOV10 and MOV10L1, two known RNA-binding helicases, in mouse testis. As expected, we find that MOV10 predominantly binds to 3' untranslated regions (UTRs) of mRNA and MOV10L1 selectively binds to Piwi-interacting RNA (piRNA) precursor transcripts. Our eCLIP method allows fast determination of major RNA species bound by various RBPs via small-scale sequencing of subclones and thus availability of qualified libraries, as a warrant for proceeding with deep sequencing. This study establishes an applicable basis for eCLIP in mammalian testis.

Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Here, we present an eCLIP protocol to determine major RNA targets of RBP candidates in testis.

增强交联免疫沉淀 (eCLIP) 方法, 有效鉴定小鼠睾丸蛋白质结合 rna

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, ...

Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes

Source: Hsieh, Y. W. et al., <a href="https://app.jove.com/v/54863">An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the electrophoretic mobility shift assay to detect protein-DNA interactions using infrared fluorescent-labeled DNA probes. When the protein binds to the DNA probe, it forms a large complex that migrates slowly on and appears as a shifted band on the gel.

用于检测转录因子-DNA 复合物的电泳迁移率变化测定

Source: Hsieh, Y. W. et al., An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides. J. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

Denaturing Urea Polyacrylamide Gel Electrophoresis for RNA Analysis: A Technique to Separate Fluorescently Labeled Phosphorylated RNA Oligonucleotides from their Non-Phosphorylated Equivalents

Source: Pillon, M. C., et al., <a href="https://www.jove.com/v/61258">Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates</a>. J. Vis. Exp. (2020)
In this video, we demonstrate a denaturing polyacrylamide gel electrophoresis for the separation of phosphorylated RNA species from their non-phosphorylated counterparts. The RNA species are fluorescently labeled for laser detection on the gel.

用于 RNA 分析的变性尿素聚丙烯酰胺凝胶电泳:一种将荧光标记的磷酸化 RNA 寡核苷酸与其非磷酸化等效核苷酸分离的技术

Source: Pillon, M. C., et al., Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates. J. Vis. Exp. (2020)
In this ...

水平凝胶电泳用于增强蛋白质 - RNA复合物的检测

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