我们感谢西蒙阿尔贝蒂协助发展本议定书。这项工作是支持由来自美国国立卫生研究院（GM25874），霍华德休斯医学研究所的Investigatorship（SL），和美国国家科学基金会predoctoral培训补助金（RH）的补助金。

<html>第1部分：准备凝胶<ol><li>组装凝胶铸造托盘。水平DNA电泳的标准设备都可以使用。对于大量样品，我们准备了一个20厘米× 24厘米，最多四个50梳子板。确保铸坯没有划痕，因为这些扭曲的印迹图像。 </li><li>创建1X TAE在1.5％琼脂糖溶液（中等或高的凝胶强度，低的平等就业机会）。体积应足以完全淹没梳齿 - 您将要载入尽可能最大限度地提高检测的样品。微波的混合物，直到琼脂糖完全溶解。 </li><li>快速添加SDS的0.1％，10％的股票。涡流来混合。如果一些琼脂糖凝固在这一步，溶解使用热板和小心，以避免沸腾。 </li><li>倒入铸造托盘的解决方案。用梳子耙出任何气泡，后来，因为它们可能干扰与传输。 </li><li>凝胶成立后，除去梳子和凝胶放入凝胶罐。完全淹没在1X TAE凝胶含有0.1％SDS。 </li></ol>第2部分：准备样品<ol><li>对于高通量分析酵母细胞裂解液中，我们开始成长快速搅拌过夜96块2毫升文化。在这种情况下，每一种文化的高表达有兴趣的蛋白质。分析低丰度蛋白质时，必须使用较大的文化卷</li><li>于2000年在室温下5分钟的离心力离心收获细胞。 </li><li>再次重悬细胞在水和离心机。 </li><li>悬浮在1毫升spheroplasting溶液。约30分钟的孵育在30 ° C（你可以通过显微镜确认spheroplasting效率）。 </li><li>在室温下5分钟800离心力离心。完全去除上清液。 </li><li>在100毫升裂解液重悬沉淀的原生质球。 </li><li>封面用胶带和2分钟的高速涡块。 </li><li> 2分钟，在4000离心力的颗粒细胞碎片。 </li><li>小心取出上清液到一个新的容器，例如，一个96孔PCR板。 </li><li>如果需要，确定的裂解液中的蛋白浓度。 </li><li> 4X样品缓冲液加入样品产生裂解液中含有1X样品缓冲。孵育在室温下5分钟。 </li><li>负载凝胶。如果需要，可以节省一半的样本量和煮沸SDS - PAGE分析。为了监测转移的程度以后，包括预染SDS - PAGE电泳标记。此外，分子量非常大的蛋白质（例如，鸡胸大肌提取物）组成的标记，可以被用来估计解决复合物的大小。 </li><li>运行在低电压（≤3 V / cm时的凝胶长度），直到染料前沿到达凝胶月底〜1厘米。这将需要几个小时。重要的是，凝胶保持冷静，否则低分子量蛋白质（例如，单体）的扩散，可以限制他们的检测。 </li></ol>第3部分：传输<ol><li>剪下一块硝化棉凝胶相同的尺寸。 </li><li> 20件GB004和GB002吸墨纸8件，切割凝胶相同的尺寸。削减GB004额外的一块，作为灯芯，它长约20厘米，比凝胶广泛。 </li><li>沉浸在1X TBS的硝化棉，灯芯，和4件GB002。 </li><li>在一个塑料容器，组装一叠论文如下：20件干GB004，干GB002 4件，然后一件预湿GB002。栈顶奠定硝化棉。 </li><li>凝胶在水中的托盘简要铸造冲洗，以除去多余的运行缓冲。然后，仔细地开始滑落到堆栈上的托盘凝胶。托盘滑出凝胶，扑灭膜用TBS必要的。额外的缓冲区，有助于防止气泡被困下的凝胶。一个移液管工程以及用于这一目的。 </li><li>后凝胶已被移动到堆栈，查深查透的气泡。如果有任何存在，解除凝胶的边缘，并重新申请缓冲区，直到气泡可以算出。 </li><li>其余三个预湿GB002件放在凝胶顶部。确保坚决整个堆栈的顶部滚动吸管彻底层之间的所有接触。 </li><li>侧翼转栈包含TBS电视台的两条高架托盘。披风预湿灯芯，灯芯两端是在TBS电视台淹没整个堆栈。 </li><li>盖上塑料轴承额外的重量（例如，一瓶500毫升的水）的一个额外的托盘组装的转栈。 </li><li>允许转让进行至少三个小时，或隔夜。 </li><li>转让后，膜可以处理标准的免疫印迹。 </li></ol> Spheroplasting解决方案 1.2米的D -山梨醇 0.5毫米氯化镁2 20毫米的Tris，pH值7.5 50 mM的生物医学工程（补充新鲜） 0.5毫克/毫升Zymolyase 100T（补充新鲜） coration：“裂解液 100毫米的Tris 7.5 50 mM氯化钠 10毫米生物医学工程（补充新鲜） 蛋白酶抑制剂（补充新鲜） 4X采样缓冲器 2X栓塞 20％的甘油 8％SDS 溴酚蓝偏好

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD - AGE Kryndushkin等人首次报道1，研究SDS耐配合物[PSI +]朊蛋白在酵母中，至今发现的广泛使用，研究朊病毒和非朊病毒聚合 2-9 。但是，转移到膜上以下的琼脂糖凝胶电泳的蛋白质是有问题的的，可以在一个扭曲的印迹图像 5的结果。此外，最常用的淹没electroblotting技术引入实际凝胶的大小限制，因此可处理的样品的数量。我们已经解决了这些问题，由用人向下毛细管转移 10，一个简单的程序，它使用一摞干印迹文件蛋白质从凝胶转移至硝酸纤维素膜上。毛细管转移防止失真，并可以轻松地处理大量凝胶。有几件事情之前要考虑使用的SDD年龄。原油样品（例如，裂解液），特定蛋白的免疫检测是必要的。 SDD - AGE不完全变性的蛋白复合体的利益，所以必须承担被检测到的蛋白（S）淀粉样地区以外的抗原表位标记。裂解液可以一般准备，因为他们将一个正常的SDS - PAGE，有两个重要的区别。首先，增加了必须小心，以防止蛋白水解降解。这里使用的部分变性条件是不够的灭活蛋白酶，也可以使靶蛋白更容易水解。至少2倍的推荐浓度使用一个完整的蛋白酶抑制剂的鸡尾酒。其次，加热的样品应避免。如果所有的单体阴性对照需要，例如，以确认高分子量物种得不到应有的共价修饰，一个孵育10分钟，在95 ° C可以使用，这将恢复最淀粉样蛋白单体。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
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screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

淀粉样蛋白聚集与大量的蛋白质错误折叠的病理学和朊病毒传染病属性的基础，这是conformationally自我模板，被认为是低等生物的有益作用的蛋白质。淀粉样已经非常困难的，由于其不溶性和结构异质性研究。然而，根据大小和洗涤剂不溶性的淀粉样聚合物分辨率可以通过半变性洗涤剂琼脂糖凝胶电泳（SDD - AGE）。这种技术是发现淀粉样蛋白的构象变体的检测和表征的广泛使用。在这里，我们展示了一种适应这种技术，有利于大规模的应用，如新的朊病毒和其他淀粉样蛋白的屏幕，它的使用。新的SDD年龄的方法使用毛细管传输提供更高的可靠性和易用性，并且可以容纳任何大小的凝胶。因此，大量的样品，准备从细胞或纯化蛋白质，可同时处理SDS不溶性标签蛋白的构象存在。

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

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

sdd的年龄是一个有用的技术检测和表征状聚合物在细胞中的淀粉样蛋白。在这里，我们展示了一个适应，使这种技术适合大规模应用。

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 ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Research

JoVE Journal

Biology

26.7K 次观看.  Whitehead Institute for Biomedical Research. sdd的年龄是一个有用的技术检测和表征状聚合物在细胞中的淀粉样蛋白。在这里，我们展示了一个适应，使这种技术适合大规模应用。

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

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

变性梯度凝胶电泳（DGGE）

科研

生物学

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

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 ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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 ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

蓝色原代聚丙烯酰胺凝胶电泳免疫印迹法诱导人细胞线粒体呼吸链配合物的分析

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...