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          2-D Electrophoresis


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          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
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