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>

<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蛋白质 - RNA结合相互作用的生物化学分析提供了许多优点。这些优点包括以下内容:与需要泵将缓冲液从下腔室转移到上部的垂直设备相比，缓冲液的混合简化，可以用较大的孔产生水平凝胶，其提供增加的分析能力ger样品，水平凝胶上的电泳通常提供与垂直凝胶相比提高的RNA-蛋白复合物的分辨率，并且水平凝胶电泳提供在分析期间在多个时间点进行成像实验的能力。 另一个主要优点是水平格式适用于创建具有大量井的凝胶，提供并行分析多个样品的能力1 。该功能显着扩展了其作为分析RNA-蛋白质相互作用的生化工具的用途。例如，分析多个样品的能力有助于通过使用竞争实验15 ，k off分析15以及旨在定义结合复合物组分15的化学计量的实验来研究蛋白质-RNA复合物形成的定量参数。另外，使用a荧光RNA底物使得有可能执行多种分析，例如荧光偏振来自单个反应结合测定，将获得的定量和定性结合数据5，15。 为了从水平凝胶设置获得最佳结果，可以调整各种参数以提高复合物的成像质量。这些包括改变凝胶的丙烯酰胺百分数，以便更容易处理和增强蛋白质-RNA复合物与游离RNA的分离。然而，由于尺寸，可能难以处理低百分比的丙烯酰胺凝胶。我们发现最容易使用丙烯酰胺浓度为7.5％或更高的凝胶。此外，如果需要，可以改变电泳条件以提高蛋白质-RNA复合物的稳定性;调整电泳电压，在室温下运行凝胶ad在4°C，并调整预运行条件。 尽管有许多优点，但水平天然凝胶电泳测定也存在一些限制。例如，通过使用荧光RNA底物使其效用最大化。根据所选择的荧光团和RNA的大小，获得这样的底物可能是昂贵的。此外，水平凝胶通常远大于垂直凝胶，因此需要较大量的材料，例如聚丙烯酰胺和DEPC处理的溶液。 水平天然凝胶电泳必须被用作用于设法确定靶向临床相关RNA-蛋白质相互作用16化合物分子画面的验证工具的潜力。这样的筛选通常使用溶液生化测定来鉴定用于进一步研究的相关化合物。与垂直格式相比，水平格式的吞吐量潜力较高有机会应用天然凝胶电泳作为潜在候选人次要验证的工具。

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的功能的控制细胞命运决定。我们的在爪蟾工作确定第一结合位点对于一个Bicc1蛋白8:32核苷酸区域从Cripto的-1 mRNA的8,12的3&#39;UTR。使用RNase保护技术的组合来定义该结合位点离子和足迹测定， 体内结合实验和体外 EMSA测定8 。 纯化的非洲爪蟾 Bicc1蛋白与荧光标记的32个核苷酸RNA混合;来自Cripto-1 mRNA的Bicc1结合位点8 。在单独的反应中，将Bicc1蛋白与来自细胞周期蛋白B1 mRNA的3&#39;UTR的荧光标记的32个核苷酸RNA混合。 Bicc1不结合细胞周期蛋白B1的mRNA 8,12和该RNA作为阴性对照。每个反应的一半在垂直天然聚丙烯酰胺凝胶上分析（ 图1A ），而另一部分使用水平天然凝胶并行分析（ 图1B ）。使用Bicc1与RNA Cripto-1 RNA结合并形成特异性复合物a的任一种方法是显而易见的并且它不结合细胞周期蛋白B1阴性对照RNA。然而，当用水平凝胶分析时，Bicc1-Cripto-1复合物显着更加明显，促进了它们的检测并且允许从未结合的RNA区分蛋白质-RNA复合物。成像后，将凝胶放回凝胶装置中并再电泳三个半小时。在重新成像凝胶后，复合物进一步迁移并且仍然易于检测，表明它们的稳定性（ 图1C ）。在初始成像后继续进行电泳和分析的这种能力将是垂直凝胶形式的挑战性的，甚至是不可能的。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56031/56031fig1.jpg" /> 图1:用于分析Bicc1与Cripto1 RNA的结合的垂直和水平天然凝胶的比较。双使用32核苷酸荧光CyclinB1 RNA阴性对照或荧光32核苷酸Cripto1 RNA阳性对照进行cc1结合实验。每个反应含有0nM或250nM的SUMO-Bicc1 N-末端，并在（ A ）垂直聚丙烯酰胺天然凝胶上在4℃在120V下运行30分钟或在水平聚丙烯酰胺天然凝胶中作为重复样品进行分析（ B ）在4℃下120V下2小时或4℃下120℃下（ C ）5.5小时。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56031/56031fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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

Nanyang Technological University

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

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

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

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 ...

科研

生物化学

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

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

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 ...

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

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

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

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

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

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

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

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

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

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

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

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

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 ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

阿西尔-PEGyl交换凝胶移位测定，用于脑膜蛋白棕榈酸化定量测定

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...