该凯克基金会慷慨地为这项工作提供了资金。这种材料是基于由FAPERJ科研补助金100.039 / 2014里约热内卢州政府支持的工作 - 巴西RDM，从科学的协调高等教育人才的改进无国界奖学金88888.075416 / 2013-00，政府下巴西，高速钢，在团契号2014171659美国国家科学基金会研究生研究奖学金用于OSS，以及研究的CNPq拨款，巴西LHFDVPCH政府202011 / 2012-7是生命的西北大学的化学的接收方处理研究所博士后奖学金二等奖。

注意:此视频协议是基于一个相关联的出版物9。特定试剂，物质和必要执行在该协议中描述的所有步骤的设备都列在材料部分。准备所需的所有解决方案的食谱和信息逐项表1。 1.梯度管凝胶的浇注<ol><li>装配铸造系统<ol><li>使用火焰加热刀片或手术刀，剪20ml的血清吸管沿正交部分10厘米处的分配尖端的顶部。确保该切割表面是光滑的。保持底部件（与尖端），丢弃该顶部件。夹住吸管底部垂直件（朝下锥尖），以支持立场。 注意:不要触摸加热的金属刀片，使用正确的手柄或钳子，以免烫伤。 </li><li>切3厘米柔性管并通过拉伸其在它连接到切吸管的分配尖端。流量控制阀连接在该管的另一端。 </li><li>连结在阀的底部，使用一个管件的梯度混合器的分配尖端。保持阀打开。 </li><li>设置在磁力搅拌器上的顶部的梯度混合器。将磁条混合室中。 </li><li>梯度混合器比吸移管件的顶端高5-10厘米，以允许该凝胶通过重力倾倒的分配尖端的位置。 </li><li>测试对于去离子水的泄漏系统。如果没有发现泄漏，通过不断前吹空调里面的干燥系统。 </li><li>覆盖吸管一块用胶带的两层或更多层的顶部。通过在圆形开口的中心的带层刺穿的孔。确保穿刺是足够大的玻璃管，通过紧紧地传递。 </li><li>滑动玻璃管，其中该凝胶将被投射，通过穿刺孔，拉伸带子，以便在玻璃配合紧密。一世nsert玻璃管，以使管的底部为2mm以上的吸管底部。 </li><li>检查，看看是否玻璃管被保持并集中和一旦系统制备未接触移液器壁。该系统现在可以用于铸造。 </li></ol></li><li>铸造凝胶<ol><li>制备凝胶缓冲液，APS和考马斯溶液（溶液1-3， 表1）。 </li><li>制备12％的T（溶液4， 表1）和1％T（溶液5， 表1）分离胶溶液。添加10微升的考马斯溶液在12％T分离凝胶溶液。立即梯度混合器浇注前添加两个解决方案APS和TEMED。 注意:聚丙烯酰胺是高度神经毒性和手套必须佩戴。 </li><li>确保梯度混合器的分配阀关闭。添加12％T分离胶溶液，以从分配尖端混合室最远。打开两个腔室之间的阀门，让溶液流动我n要在下室。 </li><li>关闭阀和吸取的脱臼12％T分离凝胶溶液返回到前室中。这样做是为了避免室之间的空气的气泡。 </li><li>添加1％T分离胶溶液，以最接近于分配尖端混合室。开启磁力搅拌器。 </li><li>同时开放两梯度混合器阀，以允许要混合的分离胶溶液和流入吸管和玻璃管。 </li><li>一旦在梯度混合器分离胶解决方案的日益枯竭，解耦从混频器和耦合它油管10毫升注射器。确保管的端部保持在吸移管中的液面之上，直到注射器连接牢固。 </li><li>使用注射器从管的其余分离胶溶液轻轻推入吸管和玻璃管。让气泡进入前关闭阀门吸管。 </li><li>轻轻加入约0.25毫升的水饱和butano层L（溶液6， 表1）在玻璃管内的凝胶溶液的顶部，以密封由氧气聚合反应中的空气和变平的顶部弯月面。 </li><li>用去离子水（不使用清洁剂）立即清洁浇注设备。 </li><li>恨不得一夜之间在室温下完成凝胶聚合。 </li></ol></li><li>从设备和存储分离凝胶管<ol><li>在第二天，小心剥离胶带切断装置的顶部。 </li><li>断开吸管下的管道阀门。从松开夹紧吸管并执行步骤1.3.3。 1.3.4和。在水槽上方。 </li><li>夫妇10毫升注射器填充有去离子水的柔性管。通过管道将水推到吸管，慢慢滑动聚合凝胶出来的吸管。 </li><li>水平握住吸管，小心地固定凝胶滑出开口端。 </li><li>下面和ARO切掉多余的聚丙烯酰胺UND使用刀片玻璃管。确保创建管内的凝胶上的平滑端，通过使与玻璃管的末端齐平。 </li><li>分配从凝胶的顶侧上的任何剩余的丁醇和设置凝胶管中的未加帽的15ml离心管中。混合0.5毫升凝胶缓冲液（溶液1， 表1）用4.5ml超纯水，并添加凝胶管的溶液，二者的顶侧（玻璃管内）和底侧（离心管）的约2ml。 </li><li>覆盖离心管的全部顶侧，包括玻璃管开口，用封口膜，以防止缓冲器蒸发。凝胶可以储存在4℃下约2个星期。 </li></ol></li></ol> 2.样品制备<ol><li>权衡两个鼠标心和在用剃刀刀片培养皿剁碎它们。 </li><li>经处理的组织添加到研钵中，并添加足够的液体氮，以覆盖样品。粉碎组织用杵，增加更多的当蒸发液氮中。 </li><li>一旦组织被充分研磨，让氮气蒸发，并于4℃下加入1ml裂解缓冲液（溶液7， 表1）每100毫克组织。移动溶液放入离心管中。 </li><li>离心机在2000×g离心在4℃下5分钟。小心收集在一个新的管中的上清液并弃沉淀。量化使用二辛可宁酸法12在上清液中的蛋白质浓度，或作为优选的。注:为首选本土细胞裂解物或亚细胞组分可制得。 CN-GELFrEE与各种不同的原生样品制备兼容。 </li><li>制备的溶解缓冲液和加载溶液（溶液8和9， 表1）。 </li><li>如果样品是粒料，重悬它在50-150微升溶解缓冲液中。保持在冰上的样品15分钟。 </li><li>缓冲交换再悬浮样本或solubilizat细胞裂解产物使用30kDa的-MWCO超离心过滤器的离子的缓冲区。 </li><li>离心在4℃下将蛋白质样品和10,000 xg离心5-10分钟，或直至保留分数体积下降到约20微升。丢弃的流量通过。 </li><li>加入400微升溶解缓冲的留存率和吹打混合。离心机在4℃和10000×g离心5-10分钟，或直至保留分数下降到约20微升。丢弃的流量通过。 </li><li>重复步骤2.9两次。 </li><li>在100μl溶解缓冲液的稀释过滤的样品（约20微升）。 </li><li>加入20微升装载解决方案样片。 </li></ol> 3. CN-GELFrEE设备<ol><li>执行CN-GELFrEE分馏，通过参照一个机械车间制造设备的部件。制造特氟隆的设备部件。请参阅图1为所有必要的信息以制造零件和叔O 表2为必要的部分的一台设备的数目。 安全注意事项:穿戴合适的个人防护装备。应注意周围的高压电源，以及所有路口应正确地安装。不要触摸设备或任何相邻潮湿地区，而电压是和电泳过程中保持周围台区域干燥。此外，电源应该完全处理时，该设备和/或收集的级分关闭。 </li><li>制备阳极缓冲和阴极缓冲液（溶液10-11， 表1）并保持在4℃或在冰上。 </li><li>通过间隔，O形环和第二隔板适合凝胶管（端无凝胶）的顶端，在"三明治"时尚。 </li><li>第二间隔物后添加缓冲室（阴极），通过侧孔完全通过螺钉，添加并拧紧螺母双方。确保GLAS的顶端小号管在缓冲室的顶部开口之前，不根据它直接或过去它固定。 </li><li>通过间隔，O形环，该收集室，和一第二隔板适合凝胶管的底端。 </li><li>第二间隔物后添加缓冲室（阳极），通过侧孔完全通过螺钉，添加并拧紧螺母双方。确保该凝胶管的下端是固定的过去的缓冲室的顶部开口，靠近但不接触的后壁。 </li><li>与阴极缓冲室使用向上支撑支架用夹子固定垂直设置CN-GELFrEE设备。 </li><li>加阳极缓冲到阳极缓冲室（底部）和阴极缓冲到阴极缓冲室（顶部）。保留所有的缓冲区，在4℃。 </li><li>轻轻拍打玻璃管从玻璃管内除去气泡并检查系统的泄漏。 </li><li>确保铂电极与在腔室和两个缓冲器接触凝胶管端部浸渍在各腔室中的缓冲。 </li><li>在阴极缓冲室（顶部）的阴性和阳性对阳极缓冲室（底部）:从电源给各个香蕉插头连接电源线。 </li><li>加载使用凝胶负载尖顶部凝胶的表面，凝胶管内的样品。 </li><li>在1瓦恒定运行凝胶，以避免过热，直到红色染料前处于凝胶管的底部。 注意:在继续之前关闭电源。 </li><li>丢弃阳极和阴极缓冲器。 </li><li> Untighten由阳极缓冲室的螺母和拆卸底部垫片和腔室，保持隔离件和O形圈。放置在收集室内部的凝胶管的底端，顶端开口，而不是过去的或直接在它之下之前。推衬垫和O形环接近该收集室。 </li><li>剪下一块纤维素透析膜，并通过在阳极缓冲佛光普照补充水分吧。覆盖AP收集腔室，并使用透析膜的底部隔离件之间erture。 </li><li>组装第二间隔后阳极缓冲室，添加并拧紧螺钉和​​螺栓。 </li><li>水平放置的设备在一个冰桶。加入新鲜的阳极和阴极缓冲来缓冲室并检查系统是否泄漏。 </li><li>添加150微升阳极缓冲进入收集室。重新连接电源线到相应的香蕉插头。 </li><li>打开以3mA的恒流电源。然后染料前应该开始洗脱成在收集腔室中的缓冲。 </li><li>当整个染料前被洗脱，关闭电源，并收集在第一部分（0分钟），将其传送到一个​​低蛋白结合的离心管。 </li><li>重新填充用150微升阳极缓冲的收集室。置于冰上所有收集到的分数。 </li><li>收集以下的时间间隔后馏分的收集第一级分:2分钟，4分钟，6分钟，8分钟，10分钟，15分钟，20分钟，25分钟，30分钟，45分钟和60分钟。不占时间而电源关闭。不要忘记收集的级分之前关闭电源，并用150微升样品之间阳极缓冲液再填充收集室。再次打开电源在收集之后开始洗脱下一小部分。 </li><li>分配所述阳极和阴极缓冲液和60分钟后，以缓冲室补充新鲜的解决方案。在90分钟和120分钟收集的级分。 </li><li>改变每小时的阳极和阴极的缓冲区。 </li><li>运行一个明确的本地或与分数蓝色本地PAGE和染色它作为首选。按照制造商的说明或首选协议。 </li><li>运行还原SDS-PAGE和染色它作为首选。按照制造商的说明或首选协议。 </li><li>清理GELFrEE分数，并提交到本机质谱分析所描述的PReviously 9。 </li></ol><img alt="图1" src="/files/ftp_upload/53597/53597fig1.jpg" /> 图1:CN-GELFrEE装置 （A）GELFrEE设备零件图纸和制造的部件（B）的照片; （℃）CN-GELFrEE装置组件方案:（1）缓冲室，（2）间隔，（3）O形环，（4）凝胶管，（5）收集室，和（6）渗析膜。 <img alt="表格1" src="/files/ftp_upload/53597/53597table1.jpg" /> 表1:溶液的表。 <img alt="表2" src="/files/ftp_upload/53597/53597table2.jpg" /> 表2:GELFrEE设备部件表。

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The authors have nothing to disclose.

CN-GELFrEE是从使用作为基于分子量蛋白质分馏载体分子9阴离子和中性洗涤剂的混合物通过凝胶基质的清晰天然（CN）PAGE缓冲液系统的。 CN-GELFrEE的与天然小鼠心脏蛋白提取物中的应用产生的低复杂性的级分用分立的带宽，同时保持生物分子装配的非共价相互作用。 CN-PAGE和还原SDS-PAGE平板凝胶之间的级分的比较表明，展示在馏分大分子和蛋白链之间的二硫键断裂之间的非共价相互作用的损?...

大多数生物过程的细胞内发生的被认为是由蛋白质组件而不是单一的蛋白质1进行。因此，为了阐明在细胞中的蛋白质亚基的特定的生物作用，有必要了解在配合物2其与其他蛋白或配体的结构的相互作用。然而，研究蛋白质本身，保持它们的非共价相互作用和活性，仍然具有挑战性。一种天然蛋白质研究的缺点是一个合适的原生的分离技术，可与各种下游蛋白质分析技术兼容。因此，最近在分离技术能够表征生物分子的非共价组件兴趣急剧3上升。 蛋白质分离技术是势在必行生物化学，生物物理学以及其他各种研究。目前本地分离技术具有intrinsiÇ缺陷减少下游分析，例如低分辨率，低吞吐量，损耗沉淀，和大量初始样品的要求兼容。串联亲和纯化通常用于蛋白质相互作用研究，但它必须为每个蛋白靶单独进行，导致它与高通量分析4不相容。尺寸排阻色谱法5，与所有已提供天然分离的，但本质上是低的分辨率的技术，并且需要高的初始样品量离子亲和层析5和密度梯度分离6选择性沉淀。 可替代地，基于凝胶的技术，如蓝母语（BN）和清除母语（CN）页（或1-D或2-D）显示的高分辨率分离。此外，与其他技术中提到的对比，这两种天然PAGE分离维持宽广的r溶解性和天然构象大分子物种，包括疏水性蛋白质的法兰。这种能力进一步扩大这些方法7,8达到了蛋白质组覆盖率，并通过CN和BN-PAGE之间不同的化学反应来实现的。 CN-PAGE通常依赖于软收取清洁剂为载体分子，取代了考马斯亮蓝染料BN-PAGE。 BN-PAGE，尽管具有较高的分辨率相关联，具有警告诸如在分离的蛋白8和考马斯分子加合物的形成降低的酶活性，后者大大有损于下游MS分析9。这两种方法，但是，在传统的低回收和窄蛋白质组覆盖率由于染色和凝胶提取局限性7相关联。 对于变性蛋白的研究，也有保持高分子的溶解度，同时执行具有高蛋白质回收率高分辨率分馏几种技术并且是与D兼容iverse分离后的蛋白质的分析技术。凝胶洗脱的液体馏分包封电泳（GELFrEE）是适合所有这些特征的分级分离技术之一。此方法是在高通量自上而下蛋白质组学研究大量应用，这表明它是快速和通用性。在GELFrEE，蛋白质变性并通过管状凝胶基质分隔基于分子量，它的孔隙率可以根据样品的要求和所需的分馏结果而变化。级分洗脱在液相，因而减少，同时保持高分辨率用SDS-PAGE相关联的恢复限制。分数等分试样然后可以通过1-D PAGE对于分子量带宽选择11进行分析。有用于与天然分离技术GELFrEE相关的优点的高需求。本文中所描述的方法中，清除本地GELFrEE（CN-GELFrEE），是GELFrEE土生土长适应。为了与一个宽s兼容在其天然状态大分子pectrum，这种方法不仅依赖于软CN-PAGE化学，而且还取决于孔隙率梯度分离，这通过消除存在于不连续的凝胶系统9孔隙率之间的苛刻过渡减少蛋白质沉淀。当施加到分馏从小鼠心脏提取的蛋白质复合物，洗脱的级分显示​​高回收率，得到一个宽范围的分子量的高分辨率分离。此外，将所得的级分与最下游的生物化学和生物物理蛋白质分析相容。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>30% acrylamide/bis solution, 37.5:1</td><td>Bio-Rad</td><td>161-0158</td><td>This solution is neurotoxic.</td></tr><tr><td>6-aminohexanoic acid (&amp;epsilon;-aminocaproic acid)</td><td>Sigma-Aldrich</td><td>A2504</td><td>This chemical is an irritant.</td></tr><tr><td>Ammonium persulfate (APS)</td><td>Fisher Scientific</td><td>7727-54-0</td><td>This chemical is flammable, toxic, and corrosive.</td></tr><tr><td>Coomassie blue G250</td><td>Bio-Rad</td><td>161-0406</td><td></td></tr><tr><td>Dodecylmaltoside</td><td>Sigma-Aldrich</td><td>D4641</td><td>This chemical is an irritant.</td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>FLUKA</td><td>3777</td><td>This chemical is toxic.</td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA)</td><td>Fisher Scientific</td><td>BP120</td><td>This chemical is toxic.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Liquid nitrogen</td><td>Any supplier</td><td>n/a</td><td>Store liquid nitrogen in containers&lt;br/&gt; designed for low-temperature&lt;br/&gt; liquids</td></tr><tr><td>Mouse heart</td><td>Bioreclamation LLC</td><td>MSE-HEART</td><td></td></tr><tr><td>n-butanol</td><td>Fisher Scientific</td><td>71-36-3</td><td>This chemical is flammable and toxic.</td></tr><tr><td>Ponceau S</td><td>Sigma-Aldrich</td><td>BP103</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Thermo Fisher Scientific</td><td>78430</td><td>This chemical is toxic.</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>30970</td><td>This chemical is an irritant.</td></tr><tr><td>Sucrose</td><td>JTBaker</td><td>4097</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Bio-Rad</td><td>161-0800</td><td>This chemical flammable and irritant.</td></tr><tr><td>Tris-HCl</td><td>Amresco</td><td>M151</td><td></td></tr><tr><td>Trycine</td><td>Bio-Rad</td><td>161-071</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gradient mixer</td><td>Hoefer</td><td>SG15</td><td></td></tr><tr><td>Magnetic stir&amp;nbsp;</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>Magnetic bars&amp;nbsp;</td><td>Any supplier</td><td>n/a</td><td>Small enough to fit inside the gradiet mixer chambers.</td></tr><tr><td>Power supply</td><td>Bio-Rad</td><td>164-5056</td><td>Voltage up to 1,500 V</td></tr><tr><td>Refrigerated centrifugue</td><td>Eppendorf</td><td>022622552</td><td>Up to 15,000 x g</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>30 kDa-MWCO ultra centrifugal filter</td><td>Millipore</td><td>UFC503096</td><td></td></tr><tr><td>Flexible tubing</td><td>Saint-Gobain</td><td>B000FOV1J0</td><td>Approximately 1 m length</td></tr><tr><td>Ice bucket</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>Liquid/air metallic or plastic valve. (&lt;em&gt;e.g.&lt;/em&gt; aquarium air/water flow control lever valve)</td><td>Any supplier&amp;nbsp;</td><td>n/a</td><td>Aquarium valves are compatible.</td></tr><tr><td>Mortar and pestle</td><td>Any supplier</td><td>n/a</td><td></td></tr><tr><td>Native PAGE 3-12% T slab gel</td><td>Invitrogen</td><td>BN1003</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>08-757-13</td><td></td></tr><tr><td>Protein low bind tube 1.5&amp;nbsp;ml</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Razor blade</td><td>IDL Tools</td><td>TE05-091C</td><td></td></tr><tr><td>Regenerated cellulose dialysis tubing 3,500 MWCO</td><td>Fisher Scientific</td><td>21-152-9</td><td></td></tr><tr><td>SDS-PAGE slab gel for any kDa</td><td>Bio-Rad</td><td>456-9036</td><td></td></tr><tr><td>Serological pipets (25&amp;nbsp;ml)</td><td>VWR</td><td>89130-900</td><td></td></tr><tr><td>Syringe (10 ml)</td><td>Any supplier</td><td>n/a</td><td></td></tr></tbody></table>

cn-gelfree - clear native gel-eluted liquid fraction entrapment electrophoresis

蛋白质复合物进行的关键细胞功能的阵列。阐明它们的非共价相互作用和动力学是非常重要的用于理解在生物系统中复合物的作用。而生物分子组件的直接表征近年来已变得日益重要，天然分级分离技术能与下游分析技术，包括质谱兼容，是必要的，以进一步扩展这些研究。然而，该领域缺乏高通量，宽范围，高回收率分离为天然蛋白质装配方法。这里，我们提出明确天然凝胶洗脱液体馏分包封电泳（CN-GELFrEE），这对于非共价蛋白组件的新颖的分离方式。 CN-GELFrEE分离性能是由来自小鼠心脏提取物分馏证明。级分收集在2小时和显示离散频带范围从〜30到500 kDa的。一个反面观察到提高分子量带宽sistent图案，各测距〜100 kDa的。此外，通过SDS-PAGE天然组分的随后再分析表明分子量转移与蛋白质复合物的变性一致。因此，CN-GELFrEE被证明提供从一个大分子量范围上的蛋白质复合物执行高分辨率和高恢复天然分离的能力，从而提供了与下游蛋白质分析兼容的级分。

200微克从低温研磨小鼠心脏提取的蛋白质的是在本机分级分离成每150微升14级分，使用在1-12％T凝胶管的CN-GELFrEE方法。的10微升每一组分的等分试样上的CN-PAGE平板凝胶和银染色运行。与CN-GELFrEE分馏得到的分馏和分辨率的清晰描绘示于图2A。级分收集在两个小时，并在凝胶测定显示增加的分子量范围从约30至500 kDa的一致的模式。每个部分包含了种类约100 kDa的不?...

Watch this Scientific Journal Video about CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis at JoVE.com

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

CN-GELFrEE  - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

cn-gelfree-clear-native-gel-eluted-liquid-fraction-entrapment

Research

JoVE Journal

Chemistry

11.5K 次观看.  Northwestern University. This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

视频: CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

变性梯度凝胶电泳（DGGE）

科研

生物学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), 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 ...

生物化学

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

浇注和运行的一种蛋白质凝胶重用商​​业磁带

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions.
This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.

Separating Protein with SDS-PAGE: Procedure and Applications

用SDS-PAGE技术分离蛋白

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

教育

JoVE Science Education

基础生物学

细胞分子生物学中的基本方法

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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二维凝胶电泳

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

化学

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 ...

CN-GELFrEE - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

摘要

探索更多视频

变性梯度凝胶电泳（DGGE）

电泳分离蛋白质

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

浇注和运行的一种蛋白质凝胶重用商业磁带

用SDS-PAGE技术分离蛋白

二维凝胶电泳

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

CN-GELFrEE - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

摘要

探索更多视频

变性梯度凝胶电泳（DGGE）

电泳分离蛋白质

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

浇注和运行的一种蛋白质凝胶重用商​​业磁带

用SDS-PAGE技术分离蛋白

二维凝胶电泳

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

浇注和运行的一种蛋白质凝胶重用商业磁带