Name: an optimized protocol for electrophoretic mobility shift assay using infrared fluorescent dye-labeled oligonucleotides
Uploaded: 2016-11-29T13:00:00
Description: Watch this Scientific Journal Video about An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides at JoVE.com

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）?...

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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。

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			<td height="21" style="height:21px;width:332px;">30% Acrylamide/Bis Solution, 37.5:1&#160;</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:207px;">1610158</td>
			<td style="width:281px;">Acrylamide is harmful and toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">6x-His Epitope Tag Antibody (HIS.H8)</td>
			<td style="width:199px;">ThermoFisher</td>
			<td style="width:207px;">MA1-21315</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti Flag M2 antibody&#160;</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:207px;">F3165-.2MG</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Bovine Serum Albumin molecular-biology-grade</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:207px;">B9000S</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:332px;">5&#39;IRDye700-labeled DNA Oligos</td>
			<td style="width:199px;">Integrated DNA Technologies</td>
			<td style="width:207px;">Custom DNA oligo</td>
			<td style="width:281px;">These are referred as 5&#39;Dye-labeled or infrared fluorescent dye-labeled DNA oligos in the manuscript. The company will custom synthesize 5&#39; IRDye labeled DNA oligonucleotides.&#160; Requires minimum 100&#956;M scale synthesis and HPLC purification.</td>
		</tr>
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			<td height="21" style="height:21px;width:332px;">Klenow Fragment (3&#39;--&#62;5&#39; exo-)</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:207px;">M0212S</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">LightShif Poly (dI-dC)</td>
			<td style="width:199px;">ThermoFisher</td>
			<td style="width:207px;">20148E</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:332px;">Mini-PROTEAN&#160;Vertical Electrophoresis Cell&#160;</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:207px;">1658000FC&#160;</td>
			<td style="width:281px;">This is referred as a mini protein gel system in the manuscript. Any electrophoretic system can be used as long as they are clear glass plates of less than 25cm x 25cm in size.</td>
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			<td height="42" style="height:42px;width:332px;">Odyssey CLx Infrared Imaging System</td>
			<td style="width:199px;">LI-COR Biotechnology</td>
			<td style="width:207px;">Odyssey CLx Infrared Imagng System</td>
			<td style="width:281px;">This is referred as an advanced infrared imaging system in the mansucript.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Odyssey Fc Imaging System&#160;</td>
			<td style="width:199px;">LI-COR Biotechnology</td>
			<td style="width:207px;">Odyssey Fc Dual-Mode Imaging System</td>
			<td style="width:281px;">This is referred as a near-infrared fluorescent imaging system primarily for Western blots in the mansucript.</td>
		</tr>
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			<td height="42" style="height:42px;width:332px;">Image Studio software (version 4.0)</td>
			<td style="width:199px;">LI-COR Biotechnology</td>
			<td style="width:207px;">Image Studio software&#160;</td>
			<td style="width:281px;">This is referred as a particular imaging software in the mansucript.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Orange G</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:207px;">O3756-25G</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">6x Orange loading dye</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td>0.25% Orange G; 30% Glycerol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5x TBE</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td>45 mM Tris-Borate; 1 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1x TE</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td>10 mM Tris-HCl; 1 mM EDTA, pH8.0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">1x STE</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td style="width:281px;">100 mM NaCl; 10 mM Tris-HCl, pH8.0; 1 mM EDTA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">5x Binding Buffer</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td style="width:281px;">50 mM Tris-HCl, pH7.5; 50 mM NaCl; 200 mM KCl; 5 mM MgCl2; 5 mM EDTA, pH8.0; 5 mM DTT; 250 ug/ml BSA</td>
		</tr>
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</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. ...

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X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

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

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 ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

MeCP2蛋白质变异的电化学发光测定

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...