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。

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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>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		<tr height="42">
			<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>
	</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

15.5K 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

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

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 ...

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

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

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

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

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

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 ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

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, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

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

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

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. ...

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.
Metabolic profiling by 1H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop&#160;conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical 1H NMR profiling.
In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to 1H NMR classical spectra.
Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant&#160;leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

<meta charset="utf8"></head><body>Pure Shift 核磁共振:植物代谢组学的新工具

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Pure Shift 核磁共振:植物代谢组学的新工具

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...