This work was funded by a Grant from the Swiss National Science foundation (Grant nr. 31003A_143834) to JP.

注意:此协议是基于以前出版的非变性PAGE协议12。在该研究中，MxA蛋白的寡聚状态使用或Vero细胞过表达的MxA或IFN-α刺激的A549细胞表达内源性的MxA进行了评估。下面描述的协议可以用来分析除了的MxA任何蛋白质的寡聚状态。然而，可能需要进一步优化。 1.细胞裂解物的制备非变性PAGE 注意:为了分析在任一的Vero或A549细胞的人MxA蛋白的寡聚状态，收获1.0×10 6个细胞。取决于细胞类型或分析蛋白质的丰度，细胞数应调整。同样重要的是要尽快光敏碘乙酰胺加入保护裂解缓冲液从曝光。 <ol><li>每孔种0.3×10 6个 A549或Vero细胞中6良好的菜肴。保持在每孔2mL的生长培养基中的细胞（ 见表1）。过夜孵育细胞在细胞培养孵育箱（37℃，5％CO 2）。 </li><li>收获洗涤的细胞用1ml磷酸盐缓冲盐水（PBS），并通过加入0.5ml 0.25％的胰蛋白酶 - 乙二胺四乙酸（EDTA），在室温下大约5分钟，1倍溶液分离。 </li><li>一旦细胞从培养皿分离，将0.5ml生长培养基中并通过上下抽吸小心混合。 </li><li>传送每个细胞以及成1个2毫升管，并用台式离心机（5000 xg离心，4℃，5分钟）沉淀它们。 </li><li>小心取出吹打上清，而不会干扰细胞沉淀。 </li><li>通过仔细吹打细胞悬液上下洗涤细胞用1ml冰冷的PBS。 </li><li>沉淀细胞在台式离心机（5000 xg离心，4℃，5分钟）。 </li><li>仔细吹打无线除去上清液thout分离细胞沉淀。 </li><li>悬浮细胞在200μl冰冷的裂解缓冲液（ 见表1）通过上下吹打和置于冰上。 </li><li>随即，用锡纸孵育冰30分钟覆盖管保护裂解液由轻。 注:在冰上孵育30分钟后，它不再是必须保护溶解物从曝光中，由于游离硫醇基团的保护是不可逆的。 </li><li>通过离心在预冷的台式离心机（13,000 xg离心，4℃，20分钟）除去细胞碎片。 </li><li>在期间离心步骤冷室于4℃20分钟平衡在透析缓冲液（ 见表1）的透析列。使用列与切断10,000的分子量。 <ol><li>附加列一个浮动浮标，并把它们放入装有透析缓冲液的烧杯中。以确保温和搅拌，使用磁力搅拌器。不要触摸膜。 注:Dialys被列能够购买或者根据由菲亚拉和同事19中描述的协议由1.5毫升管制备。 </li></ol></li><li>从透析缓冲和浮子浮标删除的列。转移清除裂解物到准备透析柱通过移液而不触及该膜。附加列一个浮动浮标，并把它们放回装满透析缓冲液中。 </li><li>在4℃下透析含有冰冷透析缓冲液（ 表1）为至少4小时（或优选过夜）的烧杯中的溶胞产物，同时用磁力搅拌器小心搅拌。使用至少百毫升透析缓冲液200微升溶胞产物。 </li><li>的透析样品转移到1.5ml试管。在台式离心机（13,000 xg离心，4℃，20分钟）除去通过离心沉淀。为了防止寡聚蛋白复合物继续透析后，立即协议（第2节）的解离。不冻结准备裂解液。 </li></ol> 2.电泳与一些修改22之前的描述进行电泳:注意。在下面描述的协议中，使用预制梯度凝胶（4-15％梯度）。或者，该凝胶可以在实验室制备。它排除任何变性剂如SDS防止寡聚蛋白复合物的离解是非常重要的。电泳时间为人类MxA蛋白的不同寡聚状态进行了优化。然而，它可根据低聚复合物的大小以及是应该可以实现分析复杂的分离范围为其它蛋白质而异。因此，电泳的最佳时间应根据经验确定。对于待分析的低聚物的最佳分辨率的电流不超过25毫安。 <ol><li>装配在凝胶室中的非变性PAGE凝胶上。填写ŧ他内部和外部腔室预冷运转缓冲液（ 表1）。 </li><li>在每凝胶25毫安为在冷室于4℃15分钟运行前预冷运转缓冲凝胶。 </li><li>混合15微升如上制备的裂解物的4倍样品缓冲液5微升（ 表1）。不要煮沸的样本。 </li><li>装载样品15微升，并在凝胶上选择的天然蛋白质的标准。在寒冷的房间在4℃运行在25毫安凝胶4小时。 注:对于半定量分析，蛋白质定量协议（ 例如 Bradford蛋白测定23）可以确保等量每个泳道的总蛋白加载执行。 </li></ol> 3.免疫印迹注意:下面描述的是一个湿印迹系统的协议。任何印迹膜都可以使用。激活聚偏二氟乙烯（PVDF）膜在100％甲醇平衡之前在印迹的buf外汇储备。在半干印迹技术可以替代地使用，但已经为大型寡聚物进行优化。 <ol><li>拆开凝胶小心地转移到SDS缓冲液（ 表1）。 </li><li>孵育在室温下10分钟，同时轻轻地摇动。 </li><li>准备2海绵，4-纤维素滤纸片及每凝胶印迹膜。浸泡其中在印迹缓冲液（ 表1）。 </li><li>组装夹心如下（底部到顶部）:1海绵，2-纤维素滤纸片材，膜，凝胶，2-纤维素滤纸片，1海绵。 </li><li>把三明治切成印迹坦克。确保该膜面对正极而凝胶面向负极。 </li><li>填充预冷印迹缓冲器中的印迹罐。 </li><li>在90毫安吸干过夜4°C的最佳蛋白转移的结果。 </li><li>拆开夹层，并在Ponc孵育膜显现蛋白质标准淡溶液，在室温下5分钟。 </li><li>退色通过仔细洗去丽春红用去离子水，直到你可以清楚的看到蛋白质标准的带膜。 </li><li>用标记笔的标准蛋白的条带。 注:残余丽春S能与免疫干扰。为了避免这种情况，该膜可以进一步孵育在0.1M的NaOH脱色1分钟和随后的洗涤，用去离子水。 </li><li>方框与封闭缓冲液的膜（ 见表1），在室温下至少1小时或过夜，在4℃。 </li><li>通过使用针对该蛋白的抗体进行分析免疫染色可视化的目的蛋白（多个）。 注:在封闭缓冲液（ 表1）1000:人MxA蛋白用特异于人MX1的兔多克隆抗体可视稀释1。抗体溶液在4℃温育过夜。可替代地，单克隆一个NTI-MxA蛋白抗体（克隆143）可用于（数据未示出）24。 </li></ol>

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The authors have nothing to disclose.

在这里，我们描述一个简单的方法，它允许通过非变性PAGE，随后通过Western印迹分析在哺乳动物细胞中表达的蛋白质的寡聚状态的快速测定。这种方法的主要优点是，一个给定的蛋白的寡聚状态可以从没有事先的蛋白质纯化全细胞裂解物来确定。这可能是对于低聚或发挥它们的相关联功能的辅助因子蛋白重要。此外，该蛋白仍然在其天然状态，如果进一步从凝胶中提取，酶活性或其它蛋白质的功?...

蛋白质的四级结构起着许多细胞过程至关重要的作用。信号通路，基因表达，和酶的激活/停用所有依赖蛋白复合物1-4的正确组装。这个过程也被称为均聚物或杂低聚是由于不可逆的共价的或可逆的静电和疏水蛋白 - 蛋白相互作用。低聚不仅多样化的不同的细胞过程，而不增加基因组的大小，而且还提供了策略蛋白质来构建稳定的复合物是对变性和降解5更耐。在低聚缺陷对蛋白质的功能产生影响，并可能导致疾病的发展。例如，酶苯丙氨酸羟化酶形成四聚体复合物。蛋白质复合物内的一些突变可以削弱四聚体的形成，并导致该疾病苯丙酮尿症6。 7。它属于dynamin上样大GTP酶超家族，并具有在体外形成8个大寡聚结构的能力。齐聚已建议，以保护从MxA蛋白快速降解9,10。尽管许多研究小组加紧努力，作用的分子机制在很大程度上仍然难以捉摸和的MxA为它的抗病毒功能齐聚国家的作用正在辩论9,11,12。在这方面，高和同事提出，其中的MxA通过在大环状寡聚结构11的形式的病毒核蛋白相互作用而发挥其抗病毒活性的模型。然而，最近，我们证明了MxA蛋白二聚体具有抗病毒活性，流感病毒12的核蛋白进行交互。乙ASED上的MxA的晶体结构，高和同事鉴定在这对于其在体外低聚和其抗病毒功能11,13临界界面区域几个氨基酸残基。因此，为了阐明它的MxA的寡聚状态发挥抗病毒活性，我们试图建立一个简单的协议，以快速地确定在人类细胞中表达以及内源的MxAIFNα刺激之后表达的MxA界面突变体的oligmeric状态。 虽然有通常用于研究蛋白质之间的相互作用，如分割绿色荧光蛋白的许多技术（分割-GFP）互补实验14，表面等离子体共振15和荧光共振能量转移（FRET）16，它们不提供寡聚蛋白复合物的精确化学计量的信息。此特定方面的调查，技术如多角度光散射（MALS）17和分析超速离心18是非常有用的。通常，使用这些方法分析的蛋白质是纯化的蛋白质。低聚方法还可能依赖于其他细胞因子。如果这些因素都是未知的，所述分析是比较困难的。此外，一些蛋白质难以在大肠杆菌表达大肠杆菌和净化。因此，这些方法都没有来分析在蜂窝环境蛋白低聚的最佳选择。此外，这些技术需要昂贵的仪器这是不容易获得的。 非变性聚丙烯酰胺凝胶电泳（PAGE），大小排阻层析或化学交联，随后通过常规的十二烷基硫酸钠（SDS）-PAGE是用于从细胞裂解物2,19,20形成低聚物的特征的有用工具。这些方法不需要专门的设备，并且可以是容易的performed在标准实验室。我们最初评价为不变地导致非特异性聚集和MxA蛋白的沉淀的各种化学交联的协议。因此，我们接下来要测试的非变性PAGE协议。非变性PAGE中排除使用SDS，和蛋白质的迁移取决于其天然电荷。蓝变性PAGE使用考马斯亮蓝G250加载蛋白质具有整体负电荷，类似于SDS，但不使蛋白质变性21。不幸的是，考马斯在高盐和通常包括在裂解缓冲液的二价阳离子（ 如 Mg 2+离子 ）的存在下艳蓝沉淀。根据所使用的缓冲液中，可能难以分析未经可能对所述寡聚蛋白复合物的作用的步骤进一步优化的样本。 在这里，我们提出了基于以前出版的方法22来确定齐聚一个简单的协议使用非变性PAGE蜂窝裂解物衍生的人MxA蛋白。

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:244px;">Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 mL</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">69570</td>
			<td style="width:268px;">Pre-equilibrate in dialysis buffer ( if Glycerol removal is desired) 
			Can be self-made according to Fiala et al. 2011</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:244px;">4&#8211;15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:131px;">456-1083</td>
			<td style="width:268px;">Pre-run in running buffer to adjust buffer system</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">cOmplete, Mini, EDTA-free</td>
			<td style="width:140px;">Roche&#160;</td>
			<td style="width:131px;">11836170001</td>
			<td>use 1 tablet per 50 ml</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:244px;">PBS, pH 7.4&#160; bottle a 500ml Gibco</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:244px;">Ponceau S solution</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:131px;">P7170</td>
			<td style="width:268px;">toxic! wear gloves and protect eyes!</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:244px;">NativeMark Unstained Protein Standard&#160; 50ul</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:131px;">P/N 57030</td>
			<td>load 5 ul/well</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">A549 cells</td>
			<td style="width:140px;">ATCC</td>
			<td style="width:131px;">ATCC CCL185</td>
			<td>Grow in growth medium (see Table 1)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">Vero cells</td>
			<td style="width:140px;">ATCC</td>
			<td style="width:131px;">ATCC CCL81</td>
			<td>Grow in growth medium (see Table 1)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">anti-Mx1 antibody</td>
			<td style="width:140px;">Novus Biologicals</td>
			<td style="width:131px;">H00004599_D01P</td>
			<td>Use at a 1:1000 dilution</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:244px;">ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)</td>
			<td style="width:140px;">GE-Healthcare</td>
			<td style="width:131px;">NA934V</td>
			<td>Use at a 1:10000 dilution</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:244px;">0.5% Trypsin-EDTA (1x)&#160;&#160;&#160;&#160;&#160;&#160;&#160; Life Technologies</td>
			<td style="width:140px;">Thermo Fisher</td>
			<td style="width:131px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Iodoacetamide&#160;&#160; 5g</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:131px;">I-6125</td>
			<td>stock&#160; 100mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Bromphenolblue</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">41966-029</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Pen&#160; Strep 100 x&#160;&#160;&#160;&#160; 100ml&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">15140 - 130</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Glutamax 100xStock, 100ml&#160;&#160;&#160;&#160; life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">350500-038</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Fetal Bovine Serum, Dialyzed , US Origin 500ml Gibco Lot:42G9552K</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cellulose filter paper</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:131px;">1703965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PVDV blotting&#160; membrane</td>
			<td style="width:140px;">GE-Healthcare</td>
			<td style="width:131px;">10600022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:140px;">Biosolve</td>
			<td style="width:131px;">0020092391BS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium fluoride (NaF)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">S-7920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">NP-40</td>
			<td style="width:140px;">Calbiochem</td>
			<td style="width:131px;">492015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">cOmplete, Mini, EDTA-free</td>
			<td style="width:140px;">Roche&#160;</td>
			<td style="width:131px;">11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Tween 20</td>
			<td style="width:140px;">Calbiochem</td>
			<td style="width:131px;">6555204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CHAPS 10% solution</td>
			<td style="width:140px;">Amresco</td>
			<td style="width:131px;">N907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Glycine</td>
			<td style="width:140px;">Biosolve</td>
			<td style="width:131px;">0007132391BS</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">sodium orthovanadate (Na3VO4)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">450243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Glycerol</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">G7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">b-Glycerophospate</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">G9422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Milk powder</td>
			<td style="width:140px;">Migros/Switzerland</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Methanol</td>
			<td style="width:140px;">Millipore</td>
			<td style="width:131px;">1.06009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium cloride (NaCl)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">71380</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">magnesium chloride (MgCl2)</td>
			<td style="width:140px;">Amresco</td>
			<td style="width:131px;">288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Sodium dodecyl sulphate (SDS)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium hydroxide (NaOH)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">S-8045</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis

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

使用非变性PAGE，我们分析了人野生型MxA蛋白，二聚体界面突变体的MxA（R640A）和MxA蛋白（L617D），以及从细胞裂解物12单体界面突变体的MxA（M527D）的寡聚状态。细胞在含有1％辛基苯氧基（NP-40）和碘乙酰胺，以确保蛋白质溶解和游离的巯基的保护缓冲液中裂解（参见图1）。如前所述，盐和小代谢物通过透析19除去。蛋白分离是通过非变性PAGE进行。促进有效免疫印迹，...

Watch this Scientific Journal Video about Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis at JoVE.com

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

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

使用非变性聚丙烯酰胺凝胶电泳人的MxA多亚基蛋白复合物的表征

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

characterization-multi-subunit-protein-complexes-human-mxa-using-non

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.2K 次观看.  University of Zurich. This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

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

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

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

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

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 ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

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

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

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

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

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

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

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 ...