MCV是CIB校内计划"更好生命的分子机器"（MACBET）的成员。导致这些结果的研究已经获得了西班牙萨洛德卡洛斯III研究所（PI12 / 01667至MCV）的资助，西班牙经济贸易部长（CTQ2015-66206-C2-2-R和SAF2015-72961-EXP至MCV） ），马德里区域政府（S2010 / BD-2316至MCV）和欧盟委员会（框架计划7（FP7））项目ComplexINC（MCV合同号279039）。资助者在研究设计，数据收集和分析，决定出版或准备稿件方面没有任何作用。

注意:使用前请咨询所有相关的材料安全数据表（MSDS）。本协议中使用的几种化学物质是有毒和腐蚀性的。使用苯酚进行tRNA提取时，请使用适当的做法，包括使用通风橱和个人防护装备（安全眼镜，手套，实验室外套，全长裤，闭式脚趾鞋等 ）。该方案使用无毒的核酸染色。使用替代污渍可能需要额外的预防措施，并且如果有毒和/或致癌（ 例如溴化乙锭），则染色剂的专门处置。 琼脂糖凝胶的制备<ol><li>称量2 g琼脂糖，并加入玻璃瓶中制备2％w / v琼脂糖凝胶。 </li><li>向含琼脂糖的瓶子中加入100毫升1x三硼酸盐 - 乙二胺四乙酸（EDTA）（TBE）运行缓冲液。通过在微波炉中加热熔化。以30秒的间隔旋转内容物进行混合并重复此步骤，直到琼脂糖完全溶解。使琼脂糖溶液冷却至约50-60℃。 </li><li>粘贴凝胶托盘的开口边缘以产生模具，放置适当的梳子以产生孔并将熔融的琼脂糖投入其中。避免气泡形成，使其在室温下固化。 </li><li>一旦凝固，将凝胶托盘放在电泳单元中，并用TBE缓冲液填充，直到凝胶完全被覆盖。 </li></ol> 2.制备tRNA <ol><li> 在大肠杆菌中表达 tRNA（UUU） <ol><li>早晨，使用用表达载体pET23d-EcUUU2（含有编码大肠杆菌 tRNA（UUU）的基因）转化的冷冻大肠杆菌 BL21（DE3）小瓶，连续制备低盐Luria Broth（LB）板补充100μg/ mL氨苄青霉素。倒置板并将其置于37℃的培养箱中，直到菌落出现（晚期）OON）。 </li><li>接种在5 mL LB加100μg/ mL氨苄青霉素中的一个良好生长的单个菌落，并在37℃下以220 rpm生长过夜。 </li><li>第二天早上，细胞沉淀（6,500 xg，4℃）10分钟，用5 mL新鲜LB加100μg/ mL氨苄西林替代培养基，并重新悬浮。使用该悬浮液接种200 mL LB加100μg/ mL氨苄青霉素。在37℃下以220rpm生长5小时。 </li><li>当590nm（OD 590 ）培养物的光密度达到0.6-0.8时，向培养物中加入1mM IPTG以触发tRNA（UUU）基因的表达。在37℃孵育3小时。 </li><li>通过在4℃下以6,500×g离心培养10分钟来收获细胞。 </li></ol></li><li> 裂解和提取 <ol><li>将细胞沉淀重悬于5mL裂解缓冲液（0.3M乙酸钠，10mM EDTA）中。从此以后，使用高压灭菌的缓冲液并将所有含tRNA的样品保持在4℃以防止tRNA降解。 </li><li>将1体积的酸性苯酚（pH 4.3）加入通风橱中的悬浮细胞沉淀。通过倒置混合1分钟以裂解细胞并以10,000xg（10分钟，4℃）离心。 </li><li>使用移液管收集含有可溶性tRNA（UUU）（和较小浓度的其他tRNA库）的上层水相，注意不要抽吸有机（底部）相。将底层和由有机相和细胞碎片组成的沉淀物丢弃到足够的容器中。 </li><li>通过在4℃下倒置1小时，将可溶（水）相与2.5体积的100％v / v乙醇充分混合，沉淀出tRNA。以15,000 xg（30分钟，4℃）离心混合物。 </li><li>仔细倾倒上清液，并将富含tRNA的沉淀重悬于4 mL凝胶过滤缓冲液（20 mM磷酸钠或磷酸钾，pH 7.4，0.1 mM EDTA）中。在2％w / v琼脂糖凝胶上运行5μL重悬浮的tRNA库。 注意:质量好的tRNA表现为单一条带（分子大小在50-100bp之间）。 </li></ol></li><li> 纯化tRNA（UUU） <ol><li>按照标准色谱实践12 ，将重悬浮的tRNA（UUU）加载到先前在凝胶过滤缓冲液中平衡的高分辨率制备型凝胶过滤柱（尺寸:16×600mm，床体积:120mL，分辨率范围:1,000-70,000Da） （流动相）。将流速设置为1 mL / min以洗脱样品。 注意:tRNA（UUU）峰的洗脱大约为75.5 mL。通常，可以获得培养物的0.5-1.0mg / L tRNA的产量。 </li><li>通过在2％w / v琼脂糖凝胶上电泳每个样品5 - 10μL，分析样品的代表性洗脱体积。 </li></ol></li></ol> 3.样品制备<ol><li>根据López-Estepa et al 。表达和纯化TcdA。 2 。 注意:最高可达250μMTcdA可用于研究。 </li><li>将相应量的TcdA根据表1移取到适当标记的1.5 mL离心管中。加入1.6mM 三 （2-羧乙基）膦（TCEP）（终浓度），并使用移液管轻轻混合。关闭盖子并在室温下孵育混合物5分钟。 </li><li>加入10μM三磷酸腺苷（ATP）和50mM MgCl 2 （终浓度）。用移液管混合，并在室温下孵育混合物5分钟。 </li><li>加入纯化的tRNA（UUU）（第2.3节），如表1所示 ，用移液管混合，室温孵育5分钟。 </li><li>加入最终达100μL的最终体积的磷酸盐缓冲盐水（PBS）。混合均匀并加入甘油至终浓度为10％v / v。 </li><li>旋转样品并短暂地将管内容物（10,000 x g，1 min，4°C）旋下。 </li></ol>凝胶加载和电泳发展<ol><li>将电泳单元放入装有碎冰的托盘中，以便在电泳过程中将室维持在4℃。 </li><li>小心地，将5μL每个样品移液到相应的孔中。添加合适的DNA大小标记。 </li><li>将电极插入电源单元的相应插槽。打开电源单元并将电压设置为100 V，并运行凝胶90分钟。 </li></ol> 5.凝胶染色观察tRNA蛋白质相互作用<ol><li>通过将50 mL TBE与1×合适的DNA染色液混合来制备染色溶液。通过用铝箔覆盖染色容器，避免将染色溶液暴露在阳光下。 注意:DNA污渍通常存储为10,000或20,000x浓缩的库存。 </li><li>电泳90分钟后，小心地从托盘中取出凝胶，并将其放入带有染色溶液的容器中。在室温下以低摇摆速度将其放置在摇杆上ure 30分钟</li><li>从染色容器中取出琼脂糖凝胶，小心点在纤维素组织或滤纸上，以除去多余的液体，并将其直接放在透照器上。打开紫外光以使含有tRNA的条带可视化。拍摄凝胶照片。 </li><li> 蛋白质染色（可选）。 <ol><li>放弃DNA染色溶液（根据安全程序），并用50 mL考马斯亮蓝（CBB）溶液代替。将托盘放置在室温摇杆上，过夜（&gt; 12 h）。丢弃CBB染色溶液，并用50 mL PBS替换，以除去多余的染料。在摇滚乐上消失1小时。 </li></ol></li><li>在光透照下可视化含蛋白质的tRNA复合物，并拍摄凝胶照片以与tRNA UV凝胶图片进行比较。 </li></ol>

<ol>
	<li>Scott, V., Clark, A. R., Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+retardation+assay.">The gel retardation assay.</a> Methods Mol Biol. 31, 339-347 (1994).</li><li>L&#243;pez-Estepa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+and+small-angle+X-ray+analysis+of+CsdL/TcdA+reveal+a+new+tRNA+binding+motif+in+the+MoeB/E1+superfamily.">The crystal structure and small-angle X-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily.</a> Plos One. 10 (4), e0118606 (2015).</li><li>Miyauchi, K., Kimura, S., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyclic+form+of+N6-threonylcarbamoyladenosine+as+a+widely+distributed+tRNA+hypermodification.">A cyclic form of N6-threonylcarbamoyladenosine as a widely distributed tRNA hypermodification.</a> Nat Chem Biol. 9 (2), 105-111 (2013).</li><li>Bolstad, H. M., Botelho, D. J., Wood, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+protein-protein+interactions+within+the+Cysteine+Sulfinate+Desulfinase+Fe-S+cluster+biogenesis+system.">Proteomic analysis of protein-protein interactions within the Cysteine Sulfinate Desulfinase Fe-S cluster biogenesis system.</a> J Proteome Res. 9 (10), 5358-5369 (2010).</li><li>Loiseau, L., Ollagnier-de Choudens, S., Lascoux, D., Forest, E., Fontecave, M., Barras, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+heteromeric+CsdA-CsdE+cysteine+desulfurase,+assisting+Fe-S+cluster+biogenesis+in+Escherichia+coli.">Analysis of the heteromeric CsdA-CsdE cysteine desulfurase, assisting Fe-S cluster biogenesis in Escherichia coli.</a> J Biol Chem. 280 (29), 26760-26769 (2005).</li><li>Fried, M. G., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+studies+of+the+cyclic+AMP+receptor+protein-DNA+interaction.">Equilibrium studies of the cyclic AMP receptor protein-DNA interaction.</a> J Mol Biol. 172 (3), 241-262 (1984).</li><li>Garner, M. M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions:+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system.">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9 (13), 3047-3060 (1981).</li><li>Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assays+for+RNA-protein+complexes.">Electrophoretic mobility shift assays for RNA-protein complexes.</a> Cold Spring Harb Protoc. 2014 (4), 435-440 (2014).</li><li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Yakhnin, A. V., Yakhnin, H., Babitzke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+mobility+shift+assays+to+detect+protein-RNA+interactions.">Gel mobility shift assays to detect protein-RNA interactions.</a> Methods Mol Biol. 905, 201-211 (2012).</li><li>Ream, J. A., Lewis, L. K., Lewis, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+agarose+gel+electrophoretic+mobility+shift+assay+for+quantitating+protein:+RNA+interactions.">Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.</a> Anal Biochem. 511, 36-41 (2016).</li><li>Hagel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-filtration+chromatography.">Gel-filtration chromatography.</a> Curr Protoc Mol Biol. , (2001).</li></ol>

作者没有什么可以披露的。

本文描述的蛋白质-TRNA琼脂糖凝胶阻滞测定可以以多种方式进行修饰。首先，可以减少凝胶中琼脂糖的百分比，以使蛋白质tRNA复合物的分离和可视化显着大于本文分析的（120 kDa）。第二，如果目标蛋白是不耐热的，则必须采取额外的预防措施，以确保通过将电泳装置移动到冷藏室或使用电泳室内的冰袋将温度保持在最高可接受温度以下，以立即消散产生的热量在运行期间。如果需要或可接受的样...

<html>凝胶阻滞测定1 （也称为电泳迁移率变动测定，EMSA）是设计用于研究和表征蛋白质 - 核酸相互作用的电泳方法。它允许使用纯化的组分或蛋白质（ 例如细胞裂解物）或核酸的复杂混合物（ 例如 ，tRNA）来分析蛋白质-DNA以及蛋白质 - RNA相互作用，特别是tRNA和tRNA结合蛋白质之间的相互作用池）。我们已经将凝胶阻滞测定法应用于纯化的tRNA（UUU）和TcdA（N6-磺酰基氨基甲酰基腺苷（t6A）脱水酶）之间相互作用的研究，该脱水酶使连接于A30的苏木糖氨基甲酰侧链在反密码子茎环（ASL）中环化，的tRNA到循环t 6 （ct 6 A） 2,3 。 TcdA也被称为CsdL 3 ，tcdA / csdL基因与csdA和csdE基因5形成簇，其编码半胱氨酸脱硫酶和半胱氨酸亚硫酸盐脱硫酸盐（CSD）系统的硫受体，并需要在体内维持TcdA功能3 。 凝胶阻滞测定的基本原理是，尽管游离核酸分子由于其大的负电荷而快速迁移到丙烯酰胺或琼脂糖凝胶的前部，但是相同核酸的蛋白质复合物的电泳迁移率显着降低。迁移率的降低可视化为核酸 - 蛋白质复合物中的"转移"，其作为离散条带解决。带正电荷的和较大的核酸 - 蛋白质复合物在凝胶上显示出更大的移动或降低迁移率。 由于复合体的电荷中和是普遍现象对于蛋白质 - 核酸复合物，凝胶阻滞测定可以应用于广泛的复合物和核酸类型。该方法简单，价格便宜，可在实验室内以最少的设备进行。它只需要少量的蛋白质和tRNA。这比替代生物物理技术有一个优势，通常消耗较大量的样品。 凝胶阻滞测定已广泛用于转录因子的研究。该测定已被用于分析许多不同DNA序列的转录因子的结合动力学，强度和特异性。 EMSA在这个领域确实是第一次开发6,7 。 RNA 8,9,10,11和tRNA分子的凝胶阻滞测定也用于核糖体研究并且像我们这里所说的那样分析在转录后修饰tRNA核苷的酶。 许多不同的参数影响凝胶阻滞测定的结果，例如温度，蛋白质和tRNA样品的质量以及结合强度。仔细规划和执行实验对于释放游离核酸和蛋白质结合物种的结果至关重要。在这里，我们使用编码tRNA（UUU）的合成基因克隆到启动子缺乏Shine-Dalgarno核糖体结合位点的表达载体中，导致大量tRNA2的合成。该详细方案旨在提供执行tRNA凝胶阻滞实验的明确指导，同时避免常见错误。

<table border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">E. coli BL21(DE3)</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:119px;">70235</td>
			<td style="width:367px;">DE3 lysogen contains T7 polymerase upon IPTG induction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">pET23d</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:119px;">69748-3</td>
			<td style="width:367px;">pET23d confers resistance to ampicillin; T7 promoter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">LB Broth, Low Salt (Lennox L Broth), Granulated</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">GL1703</td>
			<td style="width:367px;">Conventional LB Broth, High Salt, can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ampicillin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">10419313</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">Incubator Shaker (Thermostated)</td>
			<td style="width:135px;">NBS</td>
			<td style="width:119px;">Excella E24&#160;</td>
			<td style="width:367px;">Any shaker incubator with temperature control can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG), &#62;=99%</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:119px;">BP1755</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium acetate, anhydrous, &#62;99%</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">B4017</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ethylenediaminetetraacetic acid (EDTA), Disodium salt, &#62;99%</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:119px;">AC327205000</td>
			<td style="width:367px;">Harmful if inhaled.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge (refrigerated)</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:119px;">5430</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge (refrigerated) rotor</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:119px;">5430/5430P</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge</td>
			<td style="width:135px;">Sorvall</td>
			<td style="width:119px;">RC5C</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge rotor</td>
			<td style="width:135px;">Sorvall</td>
			<td style="width:119px;">SLA-3000</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 &#177; 0.2</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4682</td>
			<td style="width:367px;">Acute toxicity (oral, dermal, inhalation), corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ethanol absolute for analysis, 100% v/v</td>
			<td style="width:135px;">Merck Millipore</td>
			<td style="width:119px;">100983</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium phosphate dibasic dihydrate, Na2HPO4, &#62;=99%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">71643</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Potassium dihydrogen phosphate, KH2PO4</td>
			<td style="width:135px;">Merck</td>
			<td style="width:119px;">48,730,250</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">HiLoad 16/60 Superdex 75&#160;</td>
			<td style="width:135px;">GE Healthcare</td>
			<td style="width:119px;">28989333</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Agarose</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">MB1200</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Tris [Tris(hydroxymethyl) aminomethane HCl]</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">T1513</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Boric Acid</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">B0503</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Microwave</td>
			<td style="width:135px;">Haier</td>
			<td style="width:119px;">HDA-2070M</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4706</td>
			<td style="width:367px;">Corrosive to metals and skin.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">ATP [Adenosine 5&#8242;-triphosphate disodium salt hydrate], Grade I, &#62;=99%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2383</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:433px;">Magnesium chloride (MgCl2), anhydrous, &#62;=98%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium chloride (NaCl), &#62;99%</td>
			<td style="width:135px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP358</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Potassium chloride (KCl), &#62;99%</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">P0515</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">NZYDNA Ladder VI</td>
			<td style="width:135px;">NZYtech</td>
			<td style="width:119px;">MB8901</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Power supply</td>
			<td style="width:135px;">Consort</td>
			<td style="width:119px;">EV261</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Electrophoresis unit</td>
			<td style="width:135px;">Real Laboratory</td>
			<td style="width:119px;">RELSMINI10</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Real Safe nucleic acid staining</td>
			<td style="width:135px;">Real Laboratory</td>
			<td style="width:119px;">RBMSAFE</td>
			<td style="width:367px;">20,000X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">UV Transilluminator (G:Box/EF)</td>
			<td style="width:135px;">Syngene</td>
			<td style="width:119px;">2216498</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Coomassie Brilliant Blue G</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0770</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Rocker (Gyro-Rocker)</td>
			<td style="width:135px;">Stuart</td>
			<td style="width:119px;">SSL3</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Mixer Vortex&#160;</td>
			<td style="width:135px;">Fisher brand</td>
			<td style="width:119px;">13214789</td>
			<td style="width:367px;"></td>
		</tr>
	</tbody>
</table>

protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function

我们展示了在大肠杆菌中表达和纯化tRNA（UUU）的方法，并且通过凝胶阻滞测定法分析tRNA（UUU）与TcdA（ N 6 -苏氨酰氨基甲酰氨基腺苷（t6A）脱水酶）的结合的分析，其使苏氨酰氨基甲酰基侧链连接到tRNA的反密码子茎环（ASL）中的A37到循环t 6 A（ct 6 A）。在具有1mM异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）的大肠杆菌中诱导编码tRNA（UUU）的合成基因的转录，诱导后24小时收获含有tRNA的细胞。使用酸性酚提取法纯化RNA级分。通过凝胶过滤色谱法获得纯tRNA，其有效地将小尺寸tRNA分子与较大的完整或片段化的核酸分离。为了分析与tRNA（UUU）结合的TcdA，将TcdA与tRNA（UUU）混合，并在4℃下在天然琼脂糖凝胶上分离。游离tRNA（UUU）迁移更快，而TcdA-tRNA（UUU）复合物经历了在凝胶染色时可观察到的移动性延迟。我们证明TcdA是一种tRNA（UUU）结合酶。这种凝胶阻滞测定可用于研究TcdA突变体以及添加剂和其他蛋白质对结合的影响。

通过在强诱导型T7启动子的控制下在大肠杆菌中表达tRNA基因可以获得大量的tRNA（UUU）。表达的tRNA（UUU）积累在细胞质中，并且在天然丰富的tRNA库中富集。使用由捕获/提取步骤和凝胶过滤/抛光步骤组成的两步​​纯化方法获得EMSA级tRNA。捕获/提取步骤使用pH 4.3苯酚来实现蛋白质，细胞碎片，DNA的同时沉淀和tRNA（和其他RNA分子）的提取。在该步骤中，可以通过在2％...

Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

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

介绍了通过琼脂糖凝胶阻滞测定技术生产tRNA（UUU）和tDNA（UUU）与酶TcdA复合物的分析方案。

蛋白质tRNA琼脂糖凝胶延迟测定分析<em&gt;ñ</em<sup&gt; 6</sup&gt; - 磺酰氨基甲酰腺苷TcdA功能

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

protein-trna-agarose-gel-retardation-assays-for-analysis-n6

Research

JoVE Journal

Biochemistry

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

9.7K 次观看.  Spanish National Research Council (CSIC). 介绍了通过琼脂糖凝胶阻滞测定技术生产tRNA（UUU）和tDNA（UUU）与酶TcdA复合物的分析方案。 

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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 ...

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. ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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 ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...