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)

78.9K 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

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

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 ...

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 ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

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 ...

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

摘要

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此视频中的章节

变性梯度凝胶电泳（DGGE）

优化的纤维蛋白凝胶珠检测血管生成的研究

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

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

代谢途径的确认和发现通过¹³ C - Proteinogenic氨基酸的标签

琼脂糖凝胶电泳分离DNA片段

DNA交联聚丙烯酰胺水凝胶的制备

粘蛋白琼脂糖凝胶电泳:蛋白质印迹高分子量糖蛋白

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

摘要

探索更多视频

此视频中的章节

变性梯度凝胶电泳（DGGE）

优化的纤维蛋白凝胶珠检测血管生成的研究

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

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

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

琼脂糖凝胶电泳分离DNA片段

DNA交联聚丙烯酰胺水凝胶的制备

粘蛋白琼脂糖凝胶电泳:蛋白质印迹高分子量糖蛋白

浇注和运行的一种蛋白质凝胶重用商业磁带

代谢途径的确认和发现通过¹³ C - Proteinogenic氨基酸的标签