Name: characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis
Uploaded: 2016-10-28T15:00:00.000+00:00
Duration: 8 min 55 s
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><tbody><tr><td>Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 ml</td><td>Thermo Fisher Scientific</td><td>69570</td><td>Pre-equilibrate in dialysis buffer (if Glycerol removal is desired)&lt;br/&gt; Can be self-made according to Fiala et al. 2011</td></tr><tr><td>4&amp;ndash;15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,</td><td>Bio-Rad</td><td>456-1083</td><td>Pre-run in running buffer to adjust buffer system</td></tr><tr><td>cOmplete, Mini, EDTA-free</td><td>Roche&amp;nbsp;</td><td>11836170001</td><td>use 1 tablet per 50 ml</td></tr><tr><td>PBS, pH 7.4&amp;nbsp; bottle a 500 ml Gibco</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>Ponceau S solution</td><td>Sigma-Aldrich</td><td>P7170</td><td>&lt;strong&gt;TOXIC&lt;/strong&gt; wear gloves and protect eyes</td></tr><tr><td>NativeMark Unstained Protein Standard&amp;nbsp; 50 &amp;micro;l</td><td>Invitrogen</td><td>P/N 57030</td><td>load 5 &amp;micro;l/well</td></tr><tr><td>A549 cells</td><td>ATCC</td><td>ATCC CCL185</td><td>Grow in growth medium (see &lt;strong&gt;Table 1&lt;/strong&gt;)</td></tr><tr><td>Vero cells</td><td>ATCC</td><td>ATCC CCL81</td><td>Grow in growth medium (see &lt;strong&gt;Table 1&lt;/strong&gt;)</td></tr><tr><td>anti-Mx1 antibody</td><td>Novus Biologicals</td><td>H00004599_D01P</td><td>Use at a 1:1,000 dilution</td></tr><tr><td>ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)</td><td>GE-Healthcare</td><td>NA934V</td><td>Use at a 1:10,000 dilution</td></tr><tr><td>0.5% Trypsin-EDTA (1x)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Life Technologies</td><td>Thermo Fisher</td><td>15400-054</td><td></td></tr><tr><td>Iodoacetamide&amp;nbsp;&amp;nbsp; 5 g</td><td>Sigma-Aldrich</td><td>I-6125</td><td>stock&amp;nbsp; 100 mM</td></tr><tr><td>Bromphenolblue</td><td>Sigma-Aldrich</td><td>B0126-25G</td><td></td></tr><tr><td>DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies</td><td>Thermo Fisher Scientific</td><td>41966-029</td><td></td></tr><tr><td>Pen&amp;nbsp; Strep 100 x&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 100ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; life technologies</td><td>Thermo Fisher Scientific</td><td>15140 - 130</td><td></td></tr><tr><td>Glutamax 100x Stock, 100 ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; life technologies</td><td>Thermo Fisher Scientific</td><td>350500-038</td><td></td></tr><tr><td>Fetal Bovine Serum, Dialyzed , US Origin 500 ml Gibco Lot:42G9552K</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>Cellulose filter paper</td><td>Bio-Rad</td><td>1703965</td><td></td></tr><tr><td>PVDF blotting&amp;nbsp; membrane</td><td>GE-Healthcare</td><td>10600022</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Biosolve</td><td>0020092391BS</td><td></td></tr><tr><td>sodium fluoride (NaF)</td><td>Sigma Aldrich</td><td>S-7920</td><td></td></tr><tr><td>NP-40</td><td>Calbiochem</td><td>492015</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-free</td><td>Roche&amp;nbsp;</td><td>11836170001</td><td></td></tr><tr><td>Tween 20</td><td>Calbiochem</td><td>6555204</td><td></td></tr><tr><td>CHAPS 10% solution</td><td>Amresco</td><td>N907</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma Aldrich</td><td>43819</td><td></td></tr><tr><td>Glycine</td><td>Biosolve</td><td>0007132391BS</td><td></td></tr><tr><td>sodium orthovanadate (Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>450243</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G7757</td><td></td></tr><tr><td>&amp;#946;-Glycerophospate</td><td>Sigma Aldrich</td><td>G9422</td><td></td></tr><tr><td>Milk powder</td><td>Migros/Switzerland</td><td></td><td></td></tr><tr><td>Methanol</td><td>Millipore</td><td>1.06009</td><td></td></tr><tr><td>sodium cloride (NaCl)</td><td>Sigma Aldrich</td><td>71380</td><td></td></tr><tr><td>magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Amresco</td><td>288</td><td></td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Sigma Aldrich</td><td>L4509</td><td></td></tr><tr><td>sodium hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>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

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

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

科研

生物化学

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 ...

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 ...

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 ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

生物学

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

粘蛋白琼脂糖凝胶电泳:蛋白质印迹高分子量糖蛋白


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

完整蛋白质复合物的化学交联和质谱分析研究多亚基蛋白质组合体的体系结构

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate

Source: Rhinesmith, T. et al., <a href="https://www.jove.com/v/55341">Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples</a>. J. Vis. Exp. (2017).
This video describes multimeric PAGE for separating native protein complexes from tissue homogenates. The technique is a hybrid of blue native-PAGE and SDS-PAGE techniques. The separated complexes can be characterized and studied for their role in cell functioning.

用于分离天然蛋白质复合物的多聚体-PAGE:一种由蓝色非变性 PAGE 和 SDS-PAGE 组成的杂化分离技术，用于从组织裂解物中分离完整的多聚体蛋白质

Source: Rhinesmith, T. et al., Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples. J. Vis. Exp. (2017).
This ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Source: Zhan, X., et al. <a href="https://www.jove.com/v/56739">Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome</a> J. Vis. Exp. (2018)
In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

二维聚丙烯酰胺凝胶电泳:一种基于电荷和分子量分析复杂蛋白质混合物中蛋白质的分离技术

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue ...