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| Introduction to two-dimensional (2-D) electrophoresis
Two-dimensional electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique sorts proteins according to two independent properties in two discrete steps: the first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights (MW). Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Thousands of different proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein is obtained.
Two-dimensional electrophoresis was first introduced by P.H. O’Farrell and J. Klose in 1975. In the original technique, the first-dimension separation was performed in carrier ampholyte-containing polyacrylamide gels cast in narrow tubes. (Under the influence of an electric current, carrier ampholytes form a pH gradient, a critical component of IEF. Sample was applied to one end of each tube gel and separated at high voltages. After IEF the gel rods were removed from their tubes, equilibrated in SDS sample buffer, and placed on vertical SDS-polyacrylamide gels for the second-dimension separation. |
The power of 2-D electrophoresis as a biochemical separation technique has been recognized virtually since its introduction. Its application, however, has become significant only in the past few years as a result of a number of developments.
 | The 2-D technique has been improved to generate 2-D maps that are superior in terms of resolution and reproducibility. This new 2-D technique, developed by A. Görg and colleagues utilizes an improved first-dimension separation method that replaces the carrier ampholyte-generated pH gradients with immobilized pH gradients (IPG) and replaces the tube gels with gel strips supported by a plastic film backing. |
 | Methods for the rapid analysis of proteins have been improved to the point that single spots eluted or transferred from single 2-D gels can be rapidly identified. Mass spectroscopic techniques have been developed that allow analysis of very small quantities of peptides and proteins. Chemical microsequencing and amino acid analysis can be performed on increasingly smaller samples. Immunochemical identification is now possible with a wide assortment of available antibodies. |
 | More-powerful, less expensive computers and software are now available, allowing routine computerized evaluations of the highly complex 2-D patterns. |
 | Data about entire genomes (or substantial fractions thereof) for a number of organisms are now available, allowing rapid identification of the gene encoding a protein separated by 2-D electrophoresis. |
 | The World Wide Web provides simple, direct access to spot pattern databases for the comparison of electrophoresis results and to genome sequence databases for assignment of sequence information. |
A large and growing application of 2-D electrophoresis is "proteome analysis." Proteome analysis is "the analysis of the PROTEin complement expressed by a genOME". The analysis involves the systematic separation, identification, and quantification of many proteins simultaneously from a single sample. 2-D electrophoresis is used in this application due to its unparalleled ability to separate thousands of proteins. 2-D electrophoresis is also unique in its ability to detect post- and cotranslational modifications, which cannot be predicted from the genome sequence.
Other applications of 2-D electrophoresis include analysis of cell differentiation, detection of disease markers, monitoring therapies, drug discovery, cancer research, purity checks, and microscale protein purification. The following sections describe methods for 2-D electrophoresis using precast IPG strips (Immobiline™ DryStrip gels) available from Amersham Biosciences. The 2-D process begins with sample preparation. Proper sample preparation is absolutely essential for a good 2-D result. The next step in the 2-D process is IPG strip rehydration. IPG strips are provided dry and must be rehydrated with the appropriate additives prior to IEF. First-dimension IEF is performed on a flatbed system at very high voltages with active temperature control. Next, strip equilibration in SDS-containing buffer prepares the sample for the second-dimension separation. Following equilibration, the strip is placed on the second-dimension gel for SDS-PAGE. The final steps are visualization and analysis of the resultant two-dimensional array of spots.
In summary, the experimental sequence for 2-D electrophoresis is:
 | 1. Sample preparation |
 | 2. IPG strip rehydration |
 | 3. IEF |
 | 4. IPG strip equilibration |
 | 5. SDS-PAGE |
 | 6. Visualization |
 | 7. Analysis |
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