Name: characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis
Uploaded: 2016-10-28T15:00:00
Description: Watch this Scientific Journal Video about Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis at JoVE.com

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?...

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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 ...

Research

JoVE Journal

Biochemistry

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

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

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

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

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

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

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

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

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 ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...