这项工作是由新加坡的学术研究理事会（ARC）[授予数量90/07]支持。我们感谢支持球菌。

为这一实验过程的完整和详细的文字协议是在分子生物学中的电流的协议。 BioRad公司网站3上可以找到详细的一步一步的组装凝胶三明治和凝胶仪器的指示。 所需设备： 玻璃板（内层和外层） 10厘米的电池：10.1 × 7.3厘米（内板），10.1 × 8.2厘米（外盘），BioRad公司 20厘米的电池：20 x 20厘米（内板），20 × 22.3厘米（外盘），BioRad公司 0.5-1.5毫米凝胶梳子和垫片 凝胶铸造立场 凝胶仪器与盖和电缆 高压电源 加热块或水浴 血清移液器和移液器援助 移液器和枪头 凝胶干燥机或扫描仪试剂和溶液： 尿素（超纯） 40％的聚丙烯酰胺溶液（29:1） 10 × TBE溶液（三硼酸盐，EDTA缓冲） 去离子水，蒸馏水 TEMED 30％（W / V）过硫酸铵溶液 0.5 × TBE溶液 甲酰胺 EDTA 二甲苯蓝 溴酚蓝 甲醇 乙醇<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>卷</td><td colspan="3" style="text-align:center;"> 50毫升 </td><td colspan="3" style="text-align:center;"> 60毫升 </td><td colspan="3" style="text-align:center;"> 10毫升 </td></tr><tr><td>丙烯酰胺浓度</td><td> 10％ </td><td> 12.5％ </td><td> 15％ </td><td> 10％ </td><td> 12.5％ </td><td> 15％ </td><td> 10％ </td><td> 12.5％ </td><td> 15％ </td></tr><tr><td> g尿素</td><td> 24 </td><td> 24 </td><td> 24 </td><td> 28.8 </td><td> 28.8 </td><td> 28.8 </td><td> 4.8 </td><td> 4.8 </td><td> 4.8 </td></tr><tr><td> ml 40％的丙烯（29:1） </td><td> 12.5 </td><td> 15.625 </td><td> 18.75 </td><td> 15 </td><td> 18.75 </td><td> 22.5 </td><td> 2.5 </td><td> 3.125 </td><td> 3.75 </td></tr><tr><td> UL 30％的APS </td><td> 166 </td><td> 166 </td><td> 166 </td><td> 199.2 </td><td> 199.2 </td><td> 199.2 </td><td> 33 </td><td> 33 </td><td> 33 </td></tr><tr><td> UL TEMED </td><td> 20 </td><td> 20 </td><td> 20 </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 10 x TBE </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 6 </td><td> 6 </td><td> 6 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="10">填补了去离子水，蒸馏水的体积</td></tr></tbody></table>表1：各种丙烯酰胺浓度和体积的需要解决单链寡核苷酸的试剂和解决方案凝胶三明治装配和凝胶的制备<ol><li>根据厂家的说明，组装凝胶和修复凝胶，凝胶注模腔3。使用0.5-1.5毫米厚的垫片。 </li><li>根据当前的协议，在分子生物学，准备适当的聚丙烯酰胺溶液或如表1所列。变性丙烯酰胺凝胶对于一个20厘米× 22厘米× 1.5毫米，60毫升的凝胶溶液为10.1 × 8.2厘米× 1毫米凝胶5毫升凝胶溶液就足够了。较大的凝胶使用时所预期的产品/乐队范围内的几个基地，然后一个较长的凝胶将解决的一个单核苷酸的差异条带。如果在超过10个核苷酸不同的预期条带，较小的凝胶将适用于解决的片段。选择适合您的分离要求的百分比聚丙烯酰胺，聚丙烯酰胺浓度较高的解决低分子量的碎片。超纯尿素和混合使用所需的丙烯酰胺量。 TBE缓冲液中加入凝胶混合，得到的终浓度为0.5-1 x TBE，并填补了去离子水，蒸馏水的体积。在微波加热20秒的解决方案，它轻轻地混合。对于较大的凝胶卷重复此步骤，直到溶液的手温暖。 </li><li>倒入凝胶，立即使用血清吸管和两个玻璃板之间的自动吸管援助。避免引入气泡。插入梳子，让凝胶聚合​​为30-60分钟。 </li></ol>成立的电泳仪和prerun凝胶<ol><li>卸载从铸造室和装配凝胶仪器以生产指示3凝胶。 </li><li>填写较低的缓冲室机智运行缓冲液（0.5-1 x，简称TBE），玻璃板将分合并后的2-3厘米缓冲区。填写上层缓冲液槽凝胶顶部运行缓冲。 </li><li>小心取出梳子和运行缓冲液冲洗加载提示使用吸管和凝胶的井。 </li><li>将凝胶系统和电缆插头盖到一个高电压电源。你可以载入你的样品之前，你必须prerun至少30分钟凝胶热凝胶和凝胶去除剩余的尿素。最佳温度应介于45-55 ° C。避免温度高于60 ° C的频段可以涂抹或玻璃板可以破解。选择不断为prerun每凝胶（15-25 W）瓦。 </li></ol>样品制备<ol><li>在此期间，准备你的样品。因此，添加适量的2 ×凝胶装载组合来样。装载组合通常含有90％甲酰胺，0.5％EDTA，0.1％二甲苯蓝，0.1％溴酚蓝。 </li><li>在采集标本之前，可以装入凝胶，样品必须在70-90 ° C之间通过加热几分钟样品热变性。 </li></ol>加载和运行的凝胶<ol><li> prerun是完成时，取下盖子，并彻底冲洗尿素浸出入井前的口袋。 </li><li>仔细将样品从底部的口袋。避免引入气泡。载入缓冲区已被应用到囊中羞涩，在运行过程中保持平等的条件。 </li><li>组装的盖子，并在不断瓦运行的凝胶，凝胶温度保持55 ° C类似的prerun。观察标记的染料迁移，染料前端，直到达到凝胶低端。运行时间是依赖于使用丙烯酰胺的百分比，离子强度的缓冲液和凝胶厚度。运行，可以持续2-4小时之间。 检查上胶的温度。凝胶温度计可以帮助监测在运行过程中适当的温度。 </li></ol>处理凝胶<ol><li>当染料前端已达到凝胶凝胶较低的缓冲室。从试验箱中取出凝胶和松开夹具。拉开间隔，并仔细拆解玻璃板。如有必要，切去上部以及含有凝胶的一部分。 </li><li>小心地转移到一盘菜凝胶，凝胶固定的解决方案（TBE加5-10％的甲醇和乙醇的浓度相同）。凝胶保留到5-10分钟的解决方案，并更改缓冲区两次。 </li><li>凝胶是准备进一步处理，采用真空凝胶干燥机或直接扫描凝胶。 </li></ol>

<ol>
	<li>Maniatis, T., Jeffrey, A., van deSande, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+length+determination+of+small+double-+and+single-stranded+DNA+molecules+by+polyacrylamide+gel+electrophoresis.">Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.</a> Biochemistry. 14, 3787-3794 (1975).</li><li>Albright, L. M., Slatko, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+polyacrylamide+gel+electrophoresis.">Denaturing polyacrylamide gel electrophoresis.</a> Curr Protoc Nucleic Acid Chem. , (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PROTEAN II xi Cell and PROTEAN II xi 2-D Cell Instruction Manual. , (2001).</li></ol>

The authors have nothing to disclose.

<ol><li>带的清晰度取决于几个因素。夏普带装样量应尽可能小，即理想之间的1-5μL。然而，到15μL可装载仍然确保接受的解决办法。样品量少，更清晰的频段量结果。 </li><li>该乐队的质量也取决于凝胶的厚度。稀释剂的凝胶，如0.75毫米，大于1.5毫米厚的凝胶显示较好的效果。 </li><li>乐队的形状也可以在凝胶加载的影响，如果样品是不能进入凝胶的口袋里同样适用。包括10％的装载缓冲甘油有助于在凝胶装载和更容?...

denaturing urea polyacrylamide gel electrophoresis (urea page)

尿素页面或变性尿素聚丙烯酰胺凝胶电泳采用6-8 M尿素，变性中学的DNA或RNA的结构，是他们根据分子量聚丙烯酰胺凝胶矩阵分离。 2至500个碱基之间的片段，如单核苷酸长度的差异，可以分开使用此方法1。样品的迁移是依赖于所选择的丙烯酰胺浓度。一个比例更高的聚丙烯酰胺解决了低分子量的碎片。尿素和温度为45-55 ° C进行组合，在凝胶运行允许非结构化的DNA或RNA分子的分离。 在一般情况下，这种方法是必需的单链DNA或RNA片段，合成或标记的寡核苷酸酶裂解反应的产品，如分析或净化。 在这个视频文章中，我们展示了如何准备和运行变性尿素聚丙烯酰胺凝胶。包括技术秘诀，除了原有的协议1,2。

Watch this Scientific Journal Video about Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE at JoVE.com

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

变性尿素聚丙烯酰胺凝胶电泳用于分离单链DNA或RNA的500个核苷酸的限制。在热变性样品和非结构化的单链结合的尿素凝胶基质内迁移，根据其分子量。

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 ...

denaturing-urea-polyacrylamide-gel-electrophoresis-urea-page

Research

JoVE Journal

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

78.3K 次观看.  Nanyang Technological University, Singapore - NTU. 变性尿素聚丙烯酰胺凝胶电泳用于分离单链DNA或RNA的500个核苷酸的限制。在热变性样品和非结构化的单链结合的尿素凝胶基质内迁移，根据其分子量。

视频: Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

变性梯度凝胶电泳（DGGE）

科研

生物学

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 ...

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 ...

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 ...

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 ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA交联聚丙烯酰胺水凝胶的制备

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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 ...

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 ...

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 ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

解剖、固定和可视化 果蝇 蛹

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...