The authors would like to acknowledge Dr. John Sheehan and Dr. Lubna Abdullah for their guidance and mentoring that were central in the completion of this work. This work was supported by funds from the National Institutes of Health (P01HL108808, UH2HL123645) and the Cystic Fibrosis Foundation Therapeutics, Inc. (EHRE07XX0). Kathryn Ramsey is supported by an NHMRC Early Career Research fellowship.

1.准备缓冲器，用于粘蛋白凝胶进行蛋白质印迹<ol><li>制备1升50×TAE（Tris-乙酸-EDTA）缓冲液。 <ol><li>在将700ml蒸馏水（DH 2 O）添加242克Tris碱（0.4 M）的57.1克冰醋酸的（称量液体）（0.2M）和14.61克乙二胺四乙酸（EDTA）（50毫米）的。 </li><li>调节pH至8.0并定容至1升卫生署2 O. </li></ol></li><li>准备10毫升10X样缓冲液。 <ol><li>准备5毫升1X TAE缓冲。要做到这一点，添加5毫升甘油（50％），25毫克的溴酚蓝（0.25％），而100毫克的十二烷基硫酸钠（SDS）（1％）的。 </li></ol></li><li>制备2升20×钠柠檬酸盐（SSC）缓冲器。 <ol><li> 1.5升的dh 2 O，加350.6克氯化钠（3M）和176.4克柠檬酸三钠（钠3 C 6 H 5 O 7）（0.3米）。调节pH至7.0，使音量2升与卫生署2 O. </li></ol></li><li> 1升1X TAE-0.1％SDS缓冲。 <ol><li> 20毫升50X TAE添加到980毫升卫生署2 O的添加1克SDS，使0.1％的SDS-TAE溶液。 </li></ol></li></ol> 2. TAE-SDS琼脂糖凝胶的制备<ol><li> （ 即 ，150毫升）中倒入足量的可微波Erlenmeyer烧瓶1×TAE-0.1％SDS缓冲液，以使5 - 6毫米厚的凝胶。加0.8％（1.2克）的琼脂糖粉末。 </li><li>在30秒的间隔间歇旋转，直到琼脂糖微波瓶中完全溶解（通常为1 - 3分钟）。在此过程中使用热手垫。小心爆发沸腾，而纷飞。 </li><li>允许琼脂糖溶液冷却为5 - 10分钟。注意:琼脂糖溶液将准备倾倒时可以在手掌被放置5秒将烧瓶底部。 </li><li>由边缘用胶带封好放入梳理位置准备电泳铸造托盘（10×15厘米）。 </li><li>在T慢慢倒入琼脂糖溶液他铸造托盘，以避免气泡的形成。删除使用一个枪头气泡。等待凝胶冷却并完全固化。一旦固化，取出梳子缓慢，以避免由吸入压扁井和从托盘的边缘移除胶带。 </li><li>将琼脂糖凝胶放入胶框，并用1X TAE-0.1％SDS缓冲液填充电泳单元，直到凝胶完全覆盖。 </li></ol> 3.样和电泳分离注意:在我们的实验室，我们通常使用来自小鼠和人类样品，包括支气管肺泡灌洗液（BALF;这两个物种），细胞洗液从人支气管上皮细胞（HBE），和人的唾液和痰样品。样品可以在6M尿素后​​收集变性或通过添加蛋白酶鸡尾酒抑制剂贮存的时间（6小时）短周期。 <ol><li>通过加入上样染料的10％至每个样品（ 即准备用于装载样品，样品和3的30微升染料微升）。通过上下吹打均匀样品。简而言之降速管在5000转，收集液滴。 </li><li>谨慎地装载样品的等体积入井。 <ol><li>预湿的提示和/或使用正位移移液管，以促进高粘度样品的载荷。慢慢地吸80 -样品-染料溶液的90％（ 即 27 - 30微升），以避免移液气泡。 </li><li>慢慢加载在孔的底部，并逐渐向上移动吸管尖。为保持连接到移液管尖的样品，取45°角和细长直上方的井或轻轻使用井的侧打破粘性粘液。不要超载井。 </li></ol></li><li>从凝胶方框电泳发电机连接的电极。确保电极正确地连接（ 即 ，插入到发生器的正输出铅丹），以允许带负电荷的粘蛋白朝着正运行<html>电极。 </li><li>运行在80伏特的凝胶90分钟为一个单梳凝胶，或60 - 75分钟为一个双梳凝胶，以防止两行之间的重叠。 </li><li>关闭发电机电源并从电源断开电极。 </li><li>小心地用一只手去除铸件托盘的任一侧从凝胶盒的凝胶，以确保凝胶保持在原位。 </li></ol> 4.减少琼脂糖凝胶进行有效的粘蛋白转移<ol><li>小心地将凝胶平在一个玻璃容器中。冲洗与卫生署2 O凝胶去除TAE-SDS缓冲的痕迹。 </li><li>通过加入200ml 20×SSC的至800ml卫生署2 O的1升4×SSC缓冲液的使200毫升的10mM二硫苏糖醇（DTT）4倍的SSC溶液中加入309毫克新鲜的DTT入200ml 4倍的SSC缓冲液中。 </li><li>用浸泡DTT-4X SSC缓冲和覆盖凝胶。轻摇20分钟（每分钟10圈）。冲洗DTT无4X SSC缓冲液凝胶。 </li></ol>"&gt; 5。真空吸墨纸大会和样品转移至硝酸纤维素膜上<ol><li>准备真空记事簿转移。 <ol><li>在尺寸比凝胶（ 即 ，10×15厘米）稍宽小心切割硝酸纤维素膜上。 </li><li>与卫生署2 O湿多孔垫，并把它光滑的正面朝上的记事簿。 </li><li>用DTT - 自由4×SSC缓冲液湿硝酸纤维素膜，并将其放置在所述多孔垫的中心。 </li><li>盖与橡胶垫圈的多孔垫，留下与硝化纤维素膜精确对准的垫圈的开口。如果多孔垫仍是可见的，仔细调整膜，以确保没有差距仍然垫圈和膜之间。 </li></ol></li><li>小心地将琼脂糖凝胶在膜的顶部。确保凝胶形式与垫圈紧密的密封和凝胶的孔被定位在膜上转让<ol><li>避免在膜和凝胶之间的气泡的形成。为了尽量减少形成气泡，喷射4×SSC中的几滴缓冲在膜和从顶部滑动凝胶底部冲洗任何形成的气泡。避免对膜动凝胶蛋白质将立即开始涂抹。 </li></ol></li><li>仔细覆盖4X SSC缓冲液凝胶。 </li><li>首先吸粘蛋白转移。 <ol><li>打开真空吸墨纸ON，并设置压力40 - 50毫巴。添加4倍SSC缓冲液几滴凝胶上每5 - 10分钟，以防止凝胶干燥。运行转印90分钟。可选:完成后，用标记定向铅笔井。 </li></ol></li><li>凝胶的处置，并使用在膜边缘的塑料镊子检索硝酸纤维素膜上。 </li></ol> 6.膜封闭和检测<ol><li>用1×磷酸盐缓冲盐水（PBS）立即冲洗膜。不要让膜干燥。 </li><li>浸没膜入50ml 3％乳的PBS上的滚装封闭缓冲液和地点克尔在室温下1小时。温育后，弃去封闭缓冲液。 </li><li>浸泡在1％牛奶的PBS初级抗体溶液膜。孵育在4℃下摇杆过夜初级抗体溶液的膜。温育后，弃去第一抗体溶液。稀释的初级抗体以1:的抗体溶液2000比封闭缓冲液。注意:对于从小鼠始发样品，我们使用UNC 222兔抗MUC5B和山羊抗 - 绿色荧光蛋白（GFP）。从人类起源的样品中，我们使用H300抗MUC5B和45M1抗MUC5AC初级抗体。 </li><li>洗膜3用PBS时间上的摇杆10分钟。 </li><li>浸泡在1％牛奶的PBS二级抗体溶液膜。稀释二级抗体以1:的抗体溶液7500比封闭缓冲液，并在室温孵育一摇杆1小时（ 例如 ，700抗兔和680抗山羊）。通过在非透明共孵育避光ntainer。丢弃后1小时的二级抗体溶液。 </li><li>洗膜3用PBS次，每次5分钟。与卫生署2 O冲洗膜去除盐分。 </li><li>根据制造商的指令的红外荧光的系统上读取该膜。 </li></ol>

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作者有没有冲突披露。

粘蛋白在该视频描述Western印迹的协议结合分子生物学用于分离和转移大分子，如DNA，以用于蛋白质检测常规技术， 即 ，免疫印迹的常规技术。同样的技术可以应用到研究复杂的糖胺聚糖，如高分子量的透明质酸18的击穿的生物学。虽然这种技术能够在广泛试验的使用，成功琼脂糖Western印迹依赖于需要细致和时间依赖性的动作步骤的多个。为了最大限度地取得成功，一些关键步骤?...

粘蛋白是由线暴露于外部环境的腔粘膜表面通常产（ 例如 ，呼吸，消化，生殖道，眼表面）以及内部器官（ 如 ，胰腺，胆囊，乳腺）。这些糖蛋白的存在下保持表面水合，并形成对病原体的物理屏障。虽然粘蛋白生产至关重要粘膜健康，粘蛋白高含沙和/或异常的粘液属性可导致管阻塞，细菌定植和慢性炎症，这可能会导致不可逆的组织损伤。事件的类似的级联在几种疾病中观察到， 例如 ，囊性纤维化1，慢性中耳炎2和宫颈阴道感染3。因此，要理解在健康和疾病粘蛋白的作用，并建立常规方案进行蛋白质鉴定是很重要的。 至今，19粘蛋白的基因已被确定和编码为大的多肽链从1200（ 例如 ，MUC1）至22,000（ 例如 ，MUC16）氨基酸。的粘蛋白基因家族可以分为两个亚型:所述膜相关粘蛋白，参与细胞信号传导和表面屏蔽，和凝​​胶形成粘蛋白，负责粘液凝胶的粘弹性性质。膜结合粘蛋白大多单体并经由疏水跨膜结构域附着于细胞表面。相反，凝胶形成粘蛋白具有这对于动态聚合物网络的形成至关重要几个血管性血友病因子（vWF）样和富含半胱氨酸的结构域。大聚糖连接到丝氨酸和整个核粘蛋白分布的苏氨酸残基。这些稠密O-连接的寡糖可以向多达分子量4的80％。分子内和分子间连接的粘蛋白单体二硫键确保粘蛋白凝胶BP网络在的完整性ķ。重糖基化和多聚化的结果是，粘蛋白是在动物世界上最大的分子间，并且不能由标准凝胶电泳使用常规的SDS-PAGE聚丙烯酰胺凝胶和标准蛋白质梯进行分析。这些方法解决与分子量大于250 kDa的低/分离蛋白质，而黏蛋白单体可在MUC16的情况下可达200​​丙二醛。然而，高分子量蛋白质梯可以用于研究小粘蛋白单体（ 即 ，MUC1）。 多种技术可用于研究粘蛋白的大小，构象和相互作用。传统上，粘蛋白的生化特性是通过在变性缓冲粘蛋白通过等密度密度梯度离心分离来完成，接着大小排阻层析和免疫检测（ 例如 ，狭线印迹法）5。动态和/或多角度光散射提供丰富的粘蛋白样品的寡聚状态信息&lt;SUP&gt; 1。此外，加上免疫检测和透射电子显微镜率区带离心通常用于确定粘蛋白6的大分子构象。质谱还用于量化粘蛋白，检测蛋白水解裂解和分析寡糖组合物1,7,8。这样的技术是昂贵的，耗时的，并且通常需要大量和/或高浓度的样品。本文中所描述的方法， 即 ，通过电泳粘蛋白的分离，是可重复的，成本低，能以高通量研究可以用于提供相对粘蛋白定量，并调查聚合物组件。然而，该测定需要可能不提供对稀有粘蛋白高亲和性，高特异性粘蛋白抗体（ 例如 ，MUC19）或某些物种（ 例如 ，猪，雪貂）。 琼脂糖免疫印迹法适用于解决各种与concentra丰富的粘蛋白样品系统蒸发散范围从50微克/毫升（ 例如 ，细胞洗涤）至5毫克/毫升（ 例如 ，痰）。此法是在20世纪90年代推出，只在少数专业的实验室进行9,10。最初，该技术有助于确定在人类呼吸道分泌物11,12粘蛋白单体的亚群，并确认在杯状细胞的低聚方法，其中包括在内质网随后在高尔基体13二聚体多聚二聚体形成的。最近，针对鼠粘蛋白多克隆抗体的产生，促进了小动物模型的研究（ 如 ，粘蛋白缺乏，βENaC，OVA挑战的小鼠模型），打开一个新的研究领域的临床前研究，旨在从去除黏液检测药物化合物肺部14-17。作为在粘蛋白生物学和新颖的产生越来越大的兴趣的结果，更具体的粘蛋白抗体，我们描述herein中的方法，以通过琼脂糖凝胶电泳，真空转移到硝化纤维素和双色红外荧光检测分离粘蛋白。

<table><tbody><tr><td>Tris Base</td><td>Sigma</td><td>T6060-1kg</td><td>1 kg</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma</td><td>ARK2183-1L</td><td>1 L</td></tr><tr><td>EDTA</td><td>Sigma</td><td>EDS-100g</td><td>100 g</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-1</td><td>1 L</td></tr><tr><td>Bromophenol Blue</td><td>Sigma</td><td>114391-5g</td><td>5 g</td></tr><tr><td>SDS (Sodium Dodecyl Sulfate)</td><td>Sigma</td><td>L6026-50g</td><td>50 g</td></tr><tr><td>20x SSC (Sodium Saline Citrate) Buffer</td><td>Promega</td><td>V4261</td><td>1 L</td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271-1</td><td>1 kg</td></tr><tr><td>Trisodium Citrate (Na&lt;sub&gt;3&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>Sigma</td><td>W302600-1kg</td><td>1 kg</td></tr><tr><td>10 mM DTT (dithiothreitol)&amp;nbsp;</td><td>Sigma</td><td>D0632</td><td>25 g</td></tr><tr><td>Milk Powder</td><td>Saco</td><td></td><td>Instant non-fat dry milk</td></tr><tr><td>Dulbecco Phosphate Buffered Saline (D-PBS)</td><td>Gibco lifetechnologies</td><td>14200-025</td><td>500 ml</td></tr><tr><td>Anti-GFP Goat Primary Antibody (mouse samples)</td><td>Rockland antibodies and assays</td><td>600-101-215</td><td>1 mg</td></tr><tr><td>UNC 222 Anti-Muc5b Rabbit Primary Antibody (mouse samples)</td><td>UNC</td><td></td><td></td></tr><tr><td>H300 Anti-MUC5B Primary Antibodies (human samples)</td><td>Santa Cruz</td><td>sc-20119</td><td>200 &amp;#956;g</td></tr><tr><td>45M1 Anti-MUC5AC Primary Antibodies (human samples)</td><td>Abcam</td><td>ab3649</td><td>100 &amp;#956;g</td></tr><tr><td>Donkey Anti-rabbit 800CW IR Dye</td><td>LI-COR Biosciences</td><td>926-32213</td><td>0.5 mg</td></tr><tr><td>Donkey Anti-mouse 680LT IR Dye</td><td>LI-COR Biosciences</td><td>926-68022</td><td>0.5 mg</td></tr><tr><td>Electrophoresis Gel Box and Casting Tray</td><td>Owl Seperation Systems</td><td></td><td></td></tr><tr><td>Power Supply Box</td><td>Biorad</td><td>Model 200/20</td><td></td></tr><tr><td>Membrane Blotting Paper</td><td>Amersham&amp;nbsp;</td><td>10600016&amp;nbsp;</td><td></td></tr><tr><td>Whatman Paper and Nitrocellulose membrane&amp;nbsp;</td><td>GE&amp;nbsp;</td><td>10 439 196</td><td></td></tr><tr><td>Boekel/Appligene Vacuum Blotter 230 v</td><td>Expotech USA</td><td>230600-2</td><td></td></tr><tr><td>Odyssey Infrared Fluorescence System</td><td>LI-COR Biosciences</td><td></td><td></td></tr></tbody></table>

mucin agarose gel electrophoresis: western blotting for high-molecular-weight glycoproteins

粘蛋白，重糖基化蛋白质衬粘膜表面，已经通过保护上皮细胞对入侵的病原体演变为先天防御的重要组成部分。这些大分子的主要作用是促进微粒收集和清除，同时促进粘膜的润滑。期间蛋白质的合成，粘蛋白经受激烈O-糖基化和多聚化，从而显着地提高这些分子的质量和尺寸。这些翻译后修饰对于粘液的粘弹性能是至关重要的。作为这些分子的复杂的生化和生物物理性质的结果，与粘蛋白的工作提供了不能由常规的蛋白质分析方法来克服许多挑战。例如，它们的高分子量防止电泳迁移通过普通的聚丙烯酰胺凝胶和它们的粘性性质导致密合性试验管。然而，调查在健康粘蛋白的作用（ 例如 ，保持粘膜完整性）和疾病（ 例如 ，高含沙，mucostasis癌症）最近获得利益和粘蛋白正在研究作为治疗靶。更好地理解的生产和粘蛋白大分子的功能可能会导致新的药物的方法， 例如 ，粘蛋白颗粒胞吐和/或粘液溶解剂的抑制剂。因此，一致和可靠的协议进行调查粘蛋白生物学是科学进步的关键。这里，我们描述的常规方法使用琼脂糖凝胶分离通过电泳粘蛋白大分子，转移蛋白成硝酸纤维素膜，并检测与特定的粘蛋白抗体信号以及红外荧光凝胶阅读器。这些技术广泛适用于确定粘蛋白定量，多聚化，并测试药理化合物对粘蛋白的影响。

我们表明以下从小鼠的肺（ 图1）在BALF中琼脂糖凝胶电泳粘蛋白表达的代表性结果。在这个例子中，我们使用了琼脂糖凝胶显示粘蛋白产生以下的IL-13治疗的Tg组织Muc5ac小鼠模型的上调。 Western印迹显示了粘蛋白的表达，其可用于多聚体或单体带信号强度（ 图2）的定量分析的视觉表示。这种方法也可以用来显示在人支气管上皮（HBE）细胞洗涤和人?...

Watch this Scientific Journal Video about Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins at JoVE.com

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

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 ...

mucin-agarose-gel-electrophoresis-western-blotting-for-high-molecular

Research

JoVE Journal

Biology

25.3K 次观看.  University of North Carolina at Chapel Hill. 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. 

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

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.

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 ...

科研

生物化学

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

免疫印迹使用Invitrogen公司NuPage NOVEX双三MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

生物学

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. ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

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 ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

仓鼠卵巢细胞的快速抗体糖工程

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

生物工程

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

聚丙烯酰胺凝胶电泳和银染色检测甘油糖

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

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 ...

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Polyacrylamide Gel Electrophoresis: An Analytical Technique to Detect Heparan Sulfates of Varying Chain Lengths Isolated from Mouse Lung Tissue

Source: LaRiviere, W. B. et al., <a href="https://www.jove.com/v/62319">Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining</a>. J. Vis. Exp. (2021)
This video demonstrates the separation procedure of heparan sulfates isolated from mouse lung tissue using polyacrylamide gel electrophoresis.

聚丙烯酰胺凝胶电泳:一种检测从小鼠肺组织中分离的不同链长硫酸乙酰肝素的分析技术

Source: LaRiviere, W. B. et al., Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (2021)
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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 ...