This work was supported by an Alfred P. Sloan Research Fellowship (to C.-F.C.) and an NIH R01 grant (5R01GM098026-05 to C.-F.C.). We thank David Crowe for access to the advanced infrared imaging system.

注:使用荧光标记的DNA探针或其它形式的标记的DNA的EMSAs分担蛋白或细胞提取物制备，蛋白质-DNA结合反应，和PAGE凝胶制备和运行（ 图1A）相同的协议。关键的区别是DNA标记程序，运行后的凝胶处理步骤和检测方法。 1.凝胶制备<ol><li>制备含有0.5×TBE（45毫摩尔Tris-硼酸，1mM EDTA中）和2.5％甘油5％非变性聚丙烯酰胺凝胶，使用微型蛋白凝胶系统（8.3厘米宽×7.3厘米长×0.75毫米厚）。 <ol><li>制备30毫升凝胶溶液4凝胶，混合5毫升30％丙烯酰胺/双（37.5:1），3 5×TBE（0.45M的Tris-硼酸，10mM EDTA的），1.5毫升50％甘油的溶液， 300微升10％过硫酸铵，TEMED 15μL，20.2毫升DDH 2 O的彻底。 注:丙烯酰胺是有毒有害，配备适当的个人防护设备处理。 </l我&gt; </ol></li><li>立即施放凝胶溶液。聚合后，敷在凝胶的透明塑料包装预先润湿0.5X TBE，并将它们存储在4℃。 </li></ol> 2.红外线荧光染料标记的探针的制备注:在制备，储存和实验保持红外荧光色素标记寡核苷酸从光尽可能远。 <ol><li>设计和秩序的寡核苷酸。 <ol><li>设计长寡核苷酸〜51聚体。 注:LIM-4 / SOX-2探头的长链寡核苷酸序列是TATCATATCTATTCTA TGATTA AATACC TATTCA TTTCAAAATCTTCTCCC。长寡核苷酸不需要PAGE或HPLC纯化。 </li><li>设计的互补短寡核苷酸〜14聚体用在5&#39;末端（5&#39;Dye）和熔化温度高于37℃，红外荧光染料修饰。 注:的短寡核苷酸序列LIM-4 / SOX-2探头是5&#39;Dye-GGGAGAAGATTTTG。 5&#39;Dye标记短寡核苷酸的最小合成规模为100纳米。因此，寡核苷酸需要HPLC纯化。 </li></ol></li><li>重悬的寡核苷酸在1×的Tris-EDTA缓冲液（TE:10mM的Tris-盐酸，pH为8.0; 1mM EDTA）中，以100μM的最终浓度。 </li><li>退火短（5&#39;Dye）和长寡核苷酸。 <ol><li>混合0.6 5&#39;Dye-短寡（100μM）的100μL，长寡（100μM）的1.2微升，和钠28.2微升氯化物的Tris-EDTA缓冲液（STE:100mM NaCl的10毫摩尔Tris盐酸，pH值8.0; 1mM EDTA）中在1.5mL管。 </li><li>放置管中5分钟沸水。关闭热源，让水与退火的寡聚体在黑暗中冷却O / N。 </li></ol></li><li>用Klenow DNA聚合酶退火5&#39;Dye-短/长寡核苷酸的5&#39;突出端补以使双链DNA探针。 <ol><li>通过混合准备反应退火长/短寡核苷酸与5&#39;Dye标签的30微升，8.5微升10X缓冲克列诺，1.7微升10毫米的dNTP，1.7克列诺的μL（3&#39; - &gt; 5&#39;外切）（5U /μL）和在0.2毫升PCR管DDH 2 O的43.1微升。 </li><li>孵育在37℃下60分钟在PCR机器。 </li><li>添加3.4微升的0.5M EDTA终止在75℃下在PCR机器进行反应和热灭活20分钟。 </li></ol></li><li>稀释STE填写的探头（0.7μM）为0.1μm的终浓度。 </li><li>在-20℃储存的寡核苷酸在黑暗中待用。寡核苷酸可以存储最多在此条件下一年。 注意:为了产生突变体的结合位点的DNA探针，长寡核苷酸的突变版本被退火以5&#39;Dye标记短的寡核苷酸，随后的5&#39;用Klenow悬填充项。已经表明，与一条链的探针标记用红外荧光染料仅具有30％的用标记与染料两条链的探针的强度。如果信号强度需要加强，两条链长的寡核苷酸在5&#39;末端标记有红外荧光染料和退火以使红外荧光染料标记的探针。 </li></ol> 3.未标记/冷探针的制备（竞争对手） 注意:互补序列（长和LongR）的长寡核苷酸退火到用于与红外荧光染料标记的探针竞争分析作出的未标记探针。 <ol><li>混合20微升100μM的长，100微米LongR寡核苷酸的20微升，并且STE 60微升的1.5毫升管中。 </li><li>把试管煮沸5分钟的水，关火源，让水与退火的寡聚体冷却过夜。 </li></ol> 4.结合反应和电泳<ol><li>通过混合50微升制备1毫升5×结合缓冲液60;的1M Tris-HCl中，pH值7.5; 5M的NaCl10μL的;的1M氯化钾，1M的5微升的 MgCl 2 200微升，10微升的0.5M的EDTA，pH值8.0; 5微升1 M DTT的;的DDH 2 O 695 10毫克/毫升BSA的25微升，并微升 注:5×结合缓冲液可以被等分并储存在-20℃。另一点要考虑的是，不同的转录因子将具有不同的修改到结合缓冲液。 </li><li>右设置结合反应之前，预运行在0.5×TBE + 2.5％甘油5％的非变性聚丙烯酰胺凝胶在80伏以除去过硫酸铵的所有痕迹30 - 60分钟，或直到当前不再随时间变化。 </li><li>设置在20微升最终体积的结合反应。 <ol><li>拌4 5×结合缓冲液100μL，80 - 200纳克纯化的蛋白A（在50％甘油， 即 ，SOX-2），0.1μM染料缀合的探针的1微升（终浓度entration:5纳米），和的DDH 2 O </li><li>可选:当需要低温探针的竞争对手，添加各种浓度的冷探头（2倍，10倍，25倍，50倍，100倍， 等等 ）。 </li><li>可选的:当蛋白质A和B之间的相互作用（ 即 ，SOX-2和LIM-4）被测试，加入80 - 200纳克纯化的蛋白B（在50％甘油， 即 ，LIM-4）。 </li><li>可选:当使用核提取物制剂和特异性结合蛋白A的要被验证，添加特定蛋白A（ 即，抗的6xHis，抗FLAG 等 ）的抗体。 </li><li>孵育R / T在黑暗15分钟。 </li></ol></li><li>加载所有的结合反应的凝胶上，并在10伏/厘米至所需的距离运行凝胶。 </li></ol> 5.成像注:将凝胶直接扫描的玻璃板用先进的红外成像系统。因此，凝胶可进一步解析和多次扫描（ 图1C和<st荣&gt; 2）。主要用于Western印迹的近红外荧光成像系统还测试扫描凝胶，但只有高级红外成像系统能够扫描具有良好的分辨率的凝胶。描述的方法是针对特定的红外成像软件，虽然也可以使用其它的软件包。 <ol><li>清洁扫描仪的床的DDH 2 O和扫描前枯井。擦拭干凝胶的玻璃板上放置装有扫描器床凝胶板。 </li><li>打开红外成像软件，并进入"捕获"选项卡。当薄钢板（1毫米）被放置在扫描仪床上，用通道的设置:700，亮度:自动，分辨率:169微米，质量:中等，聚焦偏移1.5毫米。当厚板（3毫米）被放置在扫描仪床上，用通道的设置:700，亮度:自动，分辨率:84微米，质量:中等，聚焦偏移:3.5毫米。聚焦偏置依赖于玻璃板的厚度。 </li><li>选择该凝胶扫描仪上占据的面积。点击"开始"开始扫描。 </li><li>重复电泳和另外必要时进行扫描。 </li></ol>

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The authors have nothing to disclose.

fEMSAs是调查蛋白-DNA相互作用的有效工具，并且是对DNA的放射性标记的替代方案。荧光染料诸如红外染料可商购的，并与放射性DNA标记提供更安全和更环境友好的方法。使用红外荧光染料标记的寡核苷酸EMSAs不需要后运行凝胶处理步骤，因此，节省时间和成本相对于其它DNA标记技术。的时间来检测使用放射性EMSAs频带的范围可以从30分钟至数天，这取决于测试1的DNA探针的放射性和浓度。与?...

EMSAs用于通过使用天然聚丙烯酰胺凝胶电泳（PAGE），以解决感兴趣的蛋白和含有该蛋白1的潜在目标位点的标记的DNA探针的混合物，以分析DNA和蛋白质之间的相互作用。与蛋白结合的DNA探针将与自由DNA探针下迁移较慢进行比较，因此在通过聚丙烯酰胺基质其迁移延迟。通过32 p的DNA的放射性标记已经用于检测EMSAs的主要方法。虽然放射性标记的生化研究中的应用一直是有益的，可替代DNA标记的具有可比性的灵敏度方法正在使用由于与处理放射性有关的健康和安全风险。这些替代方法包括用生物素2或洋地黄毒苷（DIG）3（两者都然后通过化学发光系统检测）的DNA的共轭，所述PAGE凝胶4的SYBR绿染色，或通过扫描凝胶5,6直接检测DNA的荧光染料结合物。 用放射性标记的DNA探针EMSAs的解决凝胶需要通过放射自显影胶片或磷光系统检测到的放射性信号1,7后运行处理。使用生物素2或地高辛共轭3 DNA探针EMSAs的凝胶还必须处理并转移到合适的膜，然后通过化学发光6检测。凝胶的SYBR绿染色需要后运行凝胶染色和荧光扫描器4。因为需要使用这些DNA标记技术EMSAs后运行凝胶处理步骤，解析的凝胶可仅一次测定。与此相反，标有荧光团的DNA探针，可以直接在由扫描仪的玻璃板内的凝胶进行检测。因此，DNA-蛋白相互作用可被监测和运行期间检测在不同时间点几次，这显著降低时间和成本。已用于EMSAs的DNA的荧光染料缀合物包括Cy3的6,8-，Cy5的6,8，荧光素9，和红外荧光染料4-6。 转录调控需要的转录因子和它们的靶基因蛋白-DNA相互作用。这些相互作用的协调从动物发育过程中的共同祖产生多样的细胞类型。无偏向前遗传筛选中鉴定的错义突变，G73E，在秀丽隐杆线虫的转录因子SOX-2的高度保守的HMG DNA结合结构域。基因突变导致AWB嗅觉神经元到AWC嗅觉神经元的细胞身份转换的分子，形态和功能水平5,10。 SOX-2通过与上下文相关的伙伴转录因子和各自相互作用差异调节AWB和AWC神经元的终末分化DNA的目标网站5,10。 SOX-2在终端AWB神经元分化合作伙伴LIM-4（LHX），而SOX-2在终端AWC神经元分化合作伙伴CEH-36（OTX / OTD）。荧光素酶报告基因分析显示，SOX-2和突变SOX-2 G73E蛋白质具有与转录辅因子的LIM-4和CEH-36协同相互作用以激活AWB和AWC神经元中表达的启动子。然而，SOX-2和突变SOX-2 G73E显示启动子的微分活化性质。调查的SOX-2和SOX-2 G73E差的DNA结合活性的分子基础，fEMSAs用这些蛋白质以及它们的潜在靶位点进行。 首先，生物信息学方法被识别生物相关的DNA的荧光素酶分析中使用的1 kb的启动子区域内的结合序列。由于多个潜在SOX-2结合位点在整个子在场，我们的重点在邻近推定CEH-36或LIM-4结合位点和各种线虫物种之间进化上保守的预测SOX-2结合位点。这些序列被删除或突变，并且随后在体内测试其活性表达中AWB或AWC神经元GFP报告转基因。通过这种方法，我们鉴定了特别需要的GFP的AWB和AWC的神经元，分别为5的表达潜力LIM-4 / SOX-2和CEH-36 / SOX-2的相邻目标位点。我们研究中的DNA中的电势差SOX-2和SOX-2使用FEMSA与所识别的LIM-4 / SOX-2和CEH-36 / SOX-2的相邻目标站点G73E的结合。我们EMSA分析表明，SOX-2 G73E不结合含LIM-4 / SOX-2相邻的靶位点（在AWB基因表达必需的）DNA探针尽可能高效率WT SOX-2一样。然而，SOX-2和SOX-2 G73E曾在结合含DNA探针没有区别CEH-36 / SOX-2djacent目标网站5,10（在AWC基因的表达需要）。我们FEMSA分析提供在影响为AWB到AWC小区标识转化表型特异的DNA结合活性的机械洞察SOX-2 G73E突变的性质。在这里，我们描述FEMSA的一个优化的协议使用纯化的6xHis-SOX-2或纯化6xHis-SOX-2含LIM-4 / SOX-2相邻的靶位点作为一个案例研究，以解决红外荧光染料标记的DNA探针G73E在一起一个重要的生物学问题。

<table><tbody><tr><td>30% Acrylamide/Bis Solution, 37.5:1&amp;nbsp;</td><td>Bio-Rad</td><td>1610158</td><td>Acrylamide is harmful and toxic.</td></tr><tr><td>6x-His Epitope Tag Antibody (HIS.H8)</td><td>ThermoFisher</td><td>MA1-21315</td><td></td></tr><tr><td>Anti-Flag M2 antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F3165-.2MG</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) molecular biology grade</td><td>New England Biolabs</td><td>B9000S</td><td></td></tr><tr><td>5&#039;IRDye700-labeled DNA Oligos</td><td>Integrated DNA Technologies</td><td>Custom DNA oligo</td><td>These are referred to as &quot;5&#039;Dye-labeled or infrared fluorescent dye-labeled DNA oligos&quot; in the manuscript. The company will custom synthesize 5&#039; IRDye labeled-DNA oligonucleotides. Requires minimum 100&amp;nbsp;&amp;mu;M scale synthesis and HPLC purification.</td></tr><tr><td>Klenow Fragment (3&#039;--&gt;5&#039; exo-)</td><td>New England Biolabs</td><td>M0212S</td><td></td></tr><tr><td>LightShif Poly (dI-dC)</td><td>ThermoFisher</td><td>20148E</td><td></td></tr><tr><td>Mini-PROTEAN&amp;nbsp;Vertical Electrophoresis Cell&amp;nbsp;</td><td>Bio-Rad</td><td>1658000FC&amp;nbsp;</td><td>This is referred to as a &#039;mini protein gel system&#039; in the manuscript. Any electrophoretic system can be used as long as they are clear glass plates of less than 25 cm x 25 cm in size.</td></tr><tr><td>Odyssey CLx Infrared Imaging System</td><td>LI-COR Biotechnology</td><td>Odyssey CLx Infrared Imagng System</td><td>This is referred to as an &#039;advanced infrared imaging system&#039; in the manuscript.</td></tr><tr><td>Odyssey Fc Imaging System&amp;nbsp;</td><td>LI-COR Biotechnology</td><td>Odyssey Fc Dual-Mode Imaging System</td><td>This is referred as a &#039;near-infrared fluorescent imaging system primarily for Western blots&#039; in the manuscript.</td></tr><tr><td>Image Studio software (version 4.0)</td><td>LI-COR Biotechnology</td><td>Image Studio software&amp;nbsp;</td><td>This is referred to as a &#039;particular imaging software&#039; in the manuscript.&amp;nbsp;</td></tr><tr><td>Orange G</td><td>Sigma-Aldrich</td><td>O3756-25G</td><td></td></tr><tr><td>6x Orange loading dye</td><td></td><td></td><td>0.25% Orange G; 30% Glycerol</td></tr><tr><td>0.5x TBE</td><td></td><td></td><td>45 mM Tris-Borate; 1 mM EDTA</td></tr><tr><td>1x TE</td><td></td><td></td><td>10 mM Tris-HCl, pH 8.0; 1 mM EDTA</td></tr><tr><td>1x STE</td><td></td><td></td><td>100 mM NaCl; 10 mM Tris-HCl, pH 8.0; 1 mM EDTA</td></tr><tr><td>5x Binding Buffer</td><td></td><td></td><td>50 mM Tris-HCl, pH 7.5; 50 mM NaCl; 200 mM KCl; 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;; 5 mM EDTA, pH 8.0; 5 mM DTT; 250 &amp;#956;g/mL BSA</td></tr></tbody></table>

an optimized protocol for electrophoretic mobility shift assay using infrared fluorescent dye-labeled oligonucleotides

电泳迁移率变动分析（EMSA）为表征的蛋白质和它们的靶DNA序列之间的相互作用的仪器工具。放射性已经在EMSAs DNA标记的主要方法。然而，最近在荧光染料和扫描方法的进步促使使用的DNA的荧光标记的作为替代的放射性为容易处理的优点，节省时间，降低成本，并提高安全性。我们最近使用的荧光EMSA（FEMSA）成功地解决一个重要的生物学问题。我们FEMSA分析提供机械洞察的错义突变，G73E的效果，在高度保守的HMG转录因子SOX-2上嗅神经元类型多样化。我们发现，突变的SOX-2蛋白G73E具体改变DNA结合活性，从而导致嗅觉神经元的身份转换。这里，我们提出一种优化和成本效益的一步一步协议使用FEMSA含有作为生物例如LIM-4 / SOX-2的相邻目标站点和纯化的SOX-2蛋白（WT和突变体SOX-2 G73E蛋白）红外荧光染料标记的寡核苷酸。

橙G样染料（6×0.12橙G在100毫升30％甘油）可被加入到结合反应装载前形象化电泳的进度。其它加载染料包括溴酚蓝将在扫描过程中检测到的，因此，用图像分析（ 图1B）产生干扰。 在EMSAs的一些情况下，尤其是当使用核提取物制剂，聚deoxyinosinic-脱氧胞苷（DI-DC）被包括在结合反应以降低蛋白质的非特异性结合的DNA ...

Watch this Scientific Journal Video about An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides at JoVE.com

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

我们在这里描述使用纯化SOX-2蛋白与红外荧光染料标记的DNA探针作为案例研究解决的重要生物学问题一起荧光电泳迁移实验（FEMSA）的优化方案。

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. ...

an-optimized-protocol-for-electrophoretic-mobility-shift-assay-using

Research

JoVE Journal

Biochemistry

15.6K 次观看.  University of Illinois at Chicago. 我们在这里描述使用纯化SOX-2蛋白与红外荧光染料标记的DNA探针作为案例研究解决的重要生物学问题一起荧光电泳迁移实验（FEMSA）的优化方案。 

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

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

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 ...

科研

生物化学

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 ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

用噻唑橙代替溴代或替代染料进行 DNA 电泳

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

使用合成寡核苷酸的 DNA 酶检测

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

作者聚焦：表征来自极端微生物和常见病原体的新型酶以了解 DNA 修复和复制

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

使用修饰的合成寡核苷酸检测核酸代谢酶

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to ...

<meta charset="utf8"></head><body>使用 OPTIR-FISH 进行细胞鉴定和代谢分析的综合方案

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

<meta charset="utf8"></head><body>作者聚焦:用于复杂环境中单细胞代谢和身份分析的集成 OPTIR-FISH

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

光学光热红外荧光原位杂交 （OPTIR-FISH）

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

生物工程

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 ...

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

摘要

探索更多视频

此视频中的章节

水平凝胶电泳用于增强蛋白质 - RNA复合物的检测

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

用噻唑橙代替溴代或替代染料进行 DNA 电泳

使用修饰的合成寡核苷酸检测核酸代谢酶

使用修饰的合成寡核苷酸检测核酸代谢酶

使用修饰的合成寡核苷酸检测核酸代谢酶

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）

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

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

摘要

探索更多视频

此视频中的章节

水平凝胶电泳用于增强蛋白质 - RNA复合物的检测

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

用噻唑橙代替溴代或替代染料进行 DNA 电泳

使用修饰的合成寡核苷酸检测核酸代谢酶

使用修饰的合成寡核苷酸检测核酸代谢酶

使用修饰的合成寡核苷酸检测核酸代谢酶

光学光热红外荧光原位杂交 （OPTIR-FISH）

光学光热红外荧光原位杂交 （OPTIR-FISH）

光学光热红外荧光原位杂交 （OPTIR-FISH）

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

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）