我们感谢为准备和染色1 - D凝胶Nicola博士Wiethölter。 这项工作是由来自德意志研究联合会（GRK 1089/project 5 ND和SM）的资助，由来自于尔根Manchot的研究奖学金基金会于Nd支持。

第1部分：二维（2 - D）凝胶电泳，用杯装<ol><li> IPG -带补液等电聚焦（IEF） <ul><li>在125μL补液盐补充水分Immobiline DryStrip凝胶，pH值6-11（7厘米）[7 M尿素，硫脲2米，4％CHAPS，50毫米hydroxyethyldisulfide和2％IPG缓冲液的pH值6-11]采用Immobiline DryStrip Reswelling托盘至少10小时。 </li><li>在IEF样品缓冲液溶解沉淀蛋白质样品[7 M尿素，硫脲2米，2％CHAPS，2％的ASB - 14，50 mM的hydroxyethyldisulfide和2％IPG缓冲液的pH值6-11]对应60-100微克每100μL蛋白。 </li><li>增溶（在室温下至少30分钟）后，申请通过阳极使用样品杯的杯装的蛋白质样品。杯的体积为100μL（集成块杯允许多达150μL）。 注意：您可以负荷较大的样品量，如果你插入一个低电压的一步，在开始聚焦协议和笔芯的杯子，而仍然是一个有在杯子里的液体膜！ </li><li>进行等电聚焦Multiphor II电泳单位在梯度模式为11.1 KVH。 </li></ul></li><li>平衡和SDS - PAGE电泳<ul><li> IEF后，IPG胶条受到还原和烷基化，每次15分钟，在摇床，平衡的解决方案中使用1％和2.5％二硫苏糖醇碘乙酰胺[50毫米Tris-HCl/pH 8.8，6​​ M尿素，30％甘油和2％SDS]。 </li><li>与H 2 O冲洗平衡条和印迹上的Whatman纸，以除去多余的的平衡缓冲液。之后浸在SDS运行缓冲液（1X）条。 </li><li>第二个方面是通过SDS - PAGE垂直电泳系统与总丙烯酰胺浓度为12％。平衡IPG胶条被放置在分离胶的顶部，并用热的琼脂糖溶液[0.5％琼脂糖凝胶电泳缓冲液含有溴酚蓝]固定。 </li><li>蛋白分离为2.5小时，在15分钟为120伏，直到染料前沿到达凝胶盒底部的80 V开始。 </li></ul></li></ol>第2部分：胶体考马斯亮CBB G - 250染色 1。染色解决方案 注：染色解决方案的编制，使用分析纯（PA）和高纯度的水，如高品质的微孔系统（Milli - Q水 ）的化学品。 <table border="0"><tbody><tr><td colspan="2">考马斯亮解决方案： </td><td>公元2000毫升H 2 O </td></tr><tr><td> 0.02％（W ​​/ V） </td><td> CBB摹- 250 </td><td> 0,4克</td></tr><tr><td> 5％（W / V） </td><td>硫酸铝（14-18）水合物</td><td> 100克</td></tr><tr><td> 10％（V / V） </td><td>乙醇（96％） </td><td> 200毫升</td></tr><tr><td> 2％（V / V） </td><td>正磷酸（85％） </td><td> 47毫升</td></tr></tbody></table> 注：编制染色液的组成部分，按照下列顺序连续此外，要保持： <ol><li>首先在Milli - Q水溶解硫酸铝</li><li>其后添加乙醇，均质，混合CBB G - 250解决方案</li><li>最近的解决方案是完全溶解后，加磷酸（酸掺入酒精媒体允许进入其胶体状态的考马斯分子聚合） </li><li>终于填补了Milli - Q水</li></ol> 注：最终的染色解决方案有一个黑暗的绿色，蓝色的外观和充满游泳周围的粒子： <ul><li>不要滤液这个解决方案！ </li><li>如果您更改编制的顺序，你会得到一个紫蓝色的解决方案，是较不敏感。 </li></ul><table border="0"><tbody><tr><td colspan="2">脱色的解决方案： </td><td>公元2000毫升H 2 O </td></tr><tr><td> 10％（V / V） </td><td>乙醇（96％） </td><td> 200毫升</td></tr><tr><td> 2％（V / V） </td><td>正磷酸（85％） </td><td> 47毫升</td></tr></tbody></table> 2。染色过程<ul><li>第二维分离后，小心取出凝胶玻璃板，他们转移到Milli - Q水填补了染色菜。 </li><li>凝胶洗净Milli - Q水水平摇床（洗衣机不足，导致敏感性差，因为SDS凝胶其余扰乱边界的蛋白质染料）10分钟三次。 </li><li>摇动考马斯亮解决方案，使用前均匀分散的胶体颗粒。 </li><li>孵育与考马斯亮解决方案覆盖上一个摇床搅拌2-12小时的凝胶。这染色产生的边际的背景，所以你可以观察到染色之间的进展。 注：10分钟后，你可以看到第一个蛋白质点出现在2个小时的孵化内，约80 ％，染色，。完成的最高水平。近100％，染色，孵育过夜建议。在某些情况下，当被染色的蛋白质的数量是巨大的和解决方案变成为一个明亮的蓝色，刷新染色解决方案是必要的。 </li><li>经过染色过程中删除的考马斯解决方案和Milli - Q水冲洗两次凝胶。 注意：只要您可以重复使用的染色溶液颗粒尚存，否则丢弃（请记住，您的实验室可能有特殊规定的废物处置）。 </li><li>删除坚持与无棉绒纸巾染色菜的染料颗粒destain为10 - 60分钟。 注意：您还可以洗Milli - Q水凝胶，但是这一点，我们只推荐用于制备凝胶，因为将有最后对凝胶，在扫描过程中会出现一个不规则的，弱的考马斯电影。 </li><li>最后Milli - Q水冲洗两次凝胶：凝胶将达到原来的厚度（酒精酸性介质中，使凝胶萎缩）和中和在水中此外增强考马斯的颜色强度。 </li></ul>第3部分：代表结果如果按照上述协议，您将获得2 - D凝胶独特的解决和染深蓝色的蛋白质斑点。甚至比2 - D凝胶，银染根据舍甫琴科等 。5，良好的灵敏度和兼容性与质谱的协议，声称，我们实现平等的染色效果。 <img src="/files/ftp_upload/1431/fig1.jpg" alt="图1" /> 图1：上述实验的最后结果。康有为的考马斯协议（一）执行强烈如银染， 舍甫琴科等 。（B）在检测后的2 - D凝胶电泳的蛋白质。 第4部分：技巧和窍门<ul><li>执行一些测试你自己的样品前处理染色法。无法解释的原因，我们有时一个感兴趣的蛋白质不能被检测到成功应用甚至当毫克。在这种情况下，我们建议准备一些系列稀释，检测灵敏度。 </li><li>如果你有低 - 凝胶检测的蛋白质（S）在一般情况下，堆放过程中的SDS - PAGE分离胶转移不成功的可能原因。您可以检查这些粘蛋白染色完整的凝胶，包括堆叠区。 </li><li>快速验证了您的等电聚焦结果，您可以直接染色IPG条，没有任何进一步的第二维分离。 </li><li>偶尔打开在染色过程中的凝胶，使他们双方染色均匀（如果你保持无动于衷凝胶，染料结合到凝胶的一面）。 </li><li>我们经验丰富的，存放在一个黑暗的瓶染色液的增加其生命周期（透明瓶至4月）。 </li><li>经过染色和记录，你可以保持几年的凝胶在冰箱中（如果您添加酸性溶液中避免由真菌污染）。 </li><li>脱色凝胶插头为胰蛋白酶消化，可以加速变暖块加热器。 </li></ul>

<ol>
	<li>Fazekas de St Groth, S., Webster, S. R. G., Datyner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+new+staining+procedures+for+quantitative+estimation+of+proteins+on+electrophoretic+strips.">Two new staining procedures for quantitative estimation of proteins on electrophoretic strips.</a> Biochim. Biophys. Acta. 71, 377-391 (1963).</li><li>Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+staining+of+proteins+in+polyacrylamide+gels+including+isoelectric+focusing+gels+with+clear+background+at+nanogram+sensitivity+using+Coomassie+Brilliant+Blue+G-250+and+R-250.">Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.</a> Electrophoresis. 9, 255-262 (1988).</li><li>Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., Orecchia, P., Zardi, L., Rigetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+Silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue Silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25, 1327-1333 (2004).</li><li>Kang, D., Gho, S. G., Suh, M., Kang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+and+Fast+Protein+Detection+with+Coomassie+Brilliant+Blue+in+Sodium+Dodecyl+Sulfate-Polyacrylamide+Gel+Electrophoresis.">Highly Sensitive and Fast Protein Detection with Coomassie Brilliant Blue in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.</a> Bull. Korean Chem. Soc. 11, 1511-1512 (2002).</li><li>Shevchenko, A., Wilm, M., Vorm, O., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+sequencing+of+proteins+silver-stained+polyacrylamide+gels.">Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.</a> Anal. Chem. 68, 850-858 (1996).</li></ol>

创新或只是另一种考马斯亮协议？ 目前存在与考马斯亮蓝染色程序的多个协议。他们大多导致轻微或重大修改Neuhoff和同事2最常用的协议之一。康有为的协议的基础上Neuhoff的公式。但它真的蛋白质组学研究的一个替代的考马斯染色的方法吗？我们将图片的两个主要问题，检测限和可用性，证据表明，它绝对是一个有益的染色协议相比其他考马斯亮协议，甚至银色和荧...

<table border="1" ><tr><td> 产品名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> 对于IEF和SDS - PAGE电泳： </td><td></td><td></td><td></td></tr><tr><td> Immobiline DryStrip凝胶</td><td> GE医疗集团</td><td> 17-6001-94 </td><td>甚至可以使用2年后到期日期。 </td></tr><tr><td> Immobiline DryStrip Reswelling托盘</td><td> GE医疗集团</td><td> 80-6371-84 </td><td>不要用有机溶剂清洗。专为7-18厘米IPG条。 </td></tr><tr><td> Immobiline DryStrip套件</td><td> GE医疗集团</td><td> 18-1004-30 </td><td>包括一个托盘，电极支架，阳极和阴极电极，对准和样品杯酒吧和样品杯。 </td></tr><tr><td>每股盈利3501 XL电源</td><td> GE医疗集团</td><td> 18-1130-05 </td><td>供应电压高达3500 V。 </td></tr><tr><td> Multiphor第二电泳单位</td><td> GE医疗集团</td><td> 18-1018-06 </td><td>移动电极使国际盛事基金在所有长度的IPG胶条（7-24厘米IPG胶条） </td></tr><tr><td> PerfectBlue凝胶体系双人小号</td><td> Peqlab </td><td> 45 - 1010 - C </td><td> SDS - PAGE电泳在10X10厘米的微型胶格式。凝胶室包括一个冷却系统。 </td></tr><tr><td> 胶体考马斯亮蓝G - 250染色： </td><td></td><td></td><td></td></tr><tr><td>染色与盖菜</td><td> VWR </td><td> 216-3412 </td><td>适合用于小型凝胶。摇床可堆叠。 </td></tr><tr><td>硫酸铝- 18 -水合物</td><td>默克公司</td><td> 1.01102.5000 </td><td>我们最好与默克公司的经验。刚上市的5公斤包装。 </td></tr><tr><td>磷酸</td><td> Prolabo </td><td> 20 624.295 </td><td>玻璃瓶出售。 </td></tr></table>

fast and sensitive colloidal coomassie g-250 staining for proteins in polyacrylamide gels

考马斯亮蓝（CBB）是常用的SDS - PAGE分离蛋白的可视化，提供一个简单的染色程序和高定量的染料。此外，它是完全兼容与质谱蛋白质鉴定。但尽管有这些优势，CBB被认为是比银或荧光染色法和较不敏感，因此很少用于分析凝胶为基础的蛋白质组学方法检测蛋白质。 已作出了一些改进原来的考马斯协议 1增加CBB灵敏度。两个重大修改，以提高转换成胶体粒子的染料分子的低丰度蛋白质的检测：在1988年，Neuhoff和他的同事们应用到的CBB G - 250基于染色溶液2 20％甲醇和硫酸铵的浓度较高，和2004年Candiano等。建立了蓝银在硫酸铵和甲醇 3在场的CBB G - 250使用磷酸。然而，所有这些修改，只允许约10毫微克蛋白质的检测。一个广泛琐细的协议，胶体考马斯染色康有为等人在2002年发表了他们关于修改Neuhoff胶体CBB染色协议的络合物质。取而代之的硫酸铵，他们用硫酸铝和甲醇毒性较低的乙醇 4取代。的新型铝为基础的染色，在康有为的研究中表现出卓越的灵敏度，可检测低为1毫微克/乐队（磷酸二）与灵敏度变化不大，对蛋白质而定。 在这里，我们展示了康有为的快速和敏感的胶体考马斯亮蓝染色分析目的蛋白的协议的应用程序。我们将说明使用二维凝胶经常在我们的工作小组进行快速和容易的协议。

Watch this Scientific Journal Video about Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels at JoVE.com

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

该视频应普及胶体考马斯亮蓝G - 250染色协议，根据康<em&gt;等。</em平均4凝胶吴蛋白质检测。染色完成后2小时内，没有任何努力。我们经常使用凝胶为基础的蛋白质组学分析的目的在康有为的协议。

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 ...

fast-sensitive-colloidal-coomassie-g-250-staining-for-proteins

Research

JoVE Journal

Biology

40.0K 次观看.  University Duesseldorf. 该视频应普及胶体考马斯亮蓝G - 250染色协议，根据康等。

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

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

对肌红蛋白鲸类检测基于金免疫层析胶体试纸条的研制

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

使用基于多反应监测的人类脑组织蛋白质的多反应监测工作流程定量蛋白质组学工作流程

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

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 ...