1. <acronym id="7zf67"></acronym>

        1. <acronym id="7zf67"></acronym>
        2. Technical Service

          location:Home > 2-D Electrophoresis

          2-D Electrophoresis

          Dateline:2017-01-20

          View count:

          Share:

          \

          Proteomics, the analysis of the complete complement of proteins in a cell, tissue, or organism (the proteome), involves the detection of the presence or absence of proteins and the direct measurement of relative protein abundances. One of the greatest challenges of proteome analysis is the reproducible fractionation of complex protein mixtures while retaining the qualitative and quantitative relationships among component proteins. Currently, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), which is capable of resolving thousands of proteins in a single run, is the primary tool of proteomics research. This section describes the various steps of a typical 2-D electrophoresis workflow, including
           
          1. Protein sample preparation
          2. First-dimension electrophoresis
          3. Second-dimension electrophoresis
          4. Gel staining and protein visualization
          5. Gel imaging and protein analysis
          6. Protein identification

          Proteome analysis is used to determine which proteins in a cell, tissue, or organism are affected by changes in conditions such as disease states or developmental stages. Protein profiles in different states of a cell or an organism can be compared to identify proteins that are qualitatively and quantitatively affected by the condition of interest. Such profiling requires superior protein separation resolution and high-throughput technologies to address the potentially large numbers of proteins.

          2-D electrophoresis can be used to resolve complex mixtures of thousands of proteins. In the first dimension, proteins are separated based on differences in isoelectric point (pI). In the second dimension, they are separated according to molecular weight. Following separation, 2-D electrophoresis gels are stained for protein visualization and analysis. In combination with computer-assisted image evaluation systems for comprehensive qualitative and quantitative examination of proteomes, this electrophoresis technique allows cataloging of proteins and comparison of data among groups of researchers.
           

          The 2-D electrophoresis workflow:

          The general workflow of a 2-D gel-based proteomics experiment is outlined below, and some of the factors affecting the way the experiment is performed are discussed.

          Protein Sample Preparation
          The method of sample preparation depends on the aim of the research and is key to the success of the experiment. Factors such as the solubilities, sizes, charges, and isoelectric points (pI) of the proteins of interest are considerations for sample preparation. Sample preparation is also important for reducing the complexity of a protein mixture. A protein fraction to be separated by 2-D PAGE must be prepared in a denaturing buffer of low ionic strength that maintains the native charges of the proteins and keeps them soluble.

          First-Dimension Separation (Isoelectric Focusing)
          Proteins are first separated on the basis of pI, the pH at which a protein carries no net charge and thus will not migrate in an electrical field. The technique is called isoelectric focusing (IEF). For 2-D PAGE, IEF is best performed in an immobilized pH gradient (IPG) gel strip, which can subsequently be directly applied onto a PAGE gel for second-dimension separation.

          Second-Dimension Separation
          In the next step of 2-D electrophoresis, an anionic surfactant such as sodium dodecyl sulfate (SDS) is typically added to impart a uniform negative charge to the proteins per unit mass and so insure uniform separation based on their molecular weights. The choice of SDS-PAGE second-dimension gel properties such as polyacrylamide percentage and gradient depends on the molecular weight (MW) range of the proteins to be separated and the size of the IPG strip used in the first dimension. The ability to run many gels at the same time under the same conditions is important for the purpose of gel-to-gel comparison. 

          2-D Gel Staining
          To visualize proteins in 2-D electrophoresis gels, the proteins must be stained or labeled. The choice of staining method is determined by several factors including desired sensitivity, linear range, ease of use, expense, and the type of imaging equipment available. 

          2-D Gel Imaging
          The ability to collect data in digital form is a major factor in making 2-D electrophoresis a practical means for collecting proteomics information. Digital gel imaging allows unprejudiced comparison of gels, the transfer of information among research groups, and cataloging of immense amounts of data. Many types of imaging devices interface with software designed specifically to collect, interpret, and compare proteomics data. 

          2-D Gel Image Analysis
          Bio-Rad's PDQuest™ software and similar image analysis software packages compare gel images, annotate protein spots, and catalog data. These software packages facilitate proteomics experiments by enabling the comparison of large 2-D electrophoresis data sets. 

          Protein Identification
          Once proteins of interest are selected by differential analysis or other criteria, the proteins can be excised from gels and identified. The ability to precisely determine MW by mass spectrometry and to search databases for peptide mass matches has made high-throughput protein identification possible
          97夜夜澡人人爽人人喊-天天噜噜噜天天拍拍爽-FREE黑人大战欧美免费-人人爽天天碰狠狠添 6080| 亚洲va在线va天堂va| 学校女厕所偷拍系列视频| 西西人体大胆瓣开下部| 免费人做人爱完整版视频| 韩国AV| 成本人动画片在线视频| 欧美A片| 欧美精品videossexohd| 伊甸园电影院| 深夜福利请备好纸巾| 亚洲欧美国产综合aV| 光棍影院在全线免费观看新版| 男女牲交过程视频播放免费| 免费三级现频在线观看视频| 免费观看完整的污视频| 中文字幕乱码免费| 亚洲欧美国产综合久久| 在线看片免费人成视频| 97高清国语自产拍| 公车被强奷短文合集| 亚洲成在线AⅤ免费视频| 亚洲欧美成AⅤ人在线观看| A级毛片高清免费视频| 亚洲 动漫 偷拍?另类 校园| 日韩在线旡码免费视频| 非洲在线观看免费视频| 老汉AV| 黄页网址大全免费观看| 国产喷水福利在线视频| 欧美精品videossexohd| 秋霞网| 樱桃成视频人APP下载| 1000视频在线播放| 国产av在在免费线观看| 日日摸夜夜添夜夜添| 最新三级电影| 国内精品自拍亚洲视频| 2019年最好看中文字字幕| 日本毛片免费韩国| 亚洲AV 中文字幕 国产 欧美| 一本大道高清在线视频| 男人将机机桶女生免费| 天天看特色高清大片视频| 国内精品福利自拍在线视频| 亚洲欧洲日产国码| 男女牲交过程视频播放免费| 亚洲综合偷拍区偷拍|