Two-Dimensional+Gel+Electrophoresis

To begin using this technique, proteins must first be separated according to intrinsic charge. This separation can be performed by dissolving the sample in a small amount of uncharged solution. This leaves denatured and dissociated polypeptide chains which can be further separated by isoelectric focusing, creating the first dimension. Polyacrylamide gel is used along with appropriate buffers, which allows each protein to migrate to its corresponding position based on its isoelectric point. To create the second dimension, electrophoresis is performed again on the sample but in a direction perpendicular to the first dimension. Proteins are then separated according to size while migrating through SDS polyacrylamide gel [1].
 * Two-Dimensional Gel Electrophoresis**
 * Basic Description:**

This technique is used to separate proteins and is more powerful than one-dimensional SDS polyacrylamide gel electrophoresis, as it can resolve up to 2000 proteins displayed in a two-dimensional map [1]. [] Two-dimensional gel electrophoresis is a technique which has been advanced over the last 50 years. This technique originated through advancements made to SDS-PAGE and other chromatography techniques. O. Smithies and M. D. Poulik first recognized in 1956 that two electrophoresis processes can yield higher resolution of complex proteins than one-dimensional separations. This discovery lead to new multidimensional chromatography and electrophoresis methods which have been perfected through much research over time [2].
 * Purpose of Technique:**
 * Origin and History**

Recent research has proved 2D-PAGE techniques to be significant in the analysis of results. Chen et al (2011) experimented with different extraction techniques for 2D-PAGE analysis of Kenaf leaf proteins. Three different extraction techniques were used: trichloroacetic acid/acetone, urea/thiourea, and phenol methods. The data shows that the phenol extraction method used in 2D-PAGE yielded the clearest results when separating proteins from Kenaf leaf tissues. This data proved to be important as other factors could be further optimized to perfect this technique and test more complex proteins [3].
 * Recent Research**

A study by Liu et al (2008) found that soil bacteria analysis could not be performed adequately using traditional gel electrophoresis techniques as these did not display a clear resolution of bacterial DNA fragments on the gel. Therefore, 2D gel electrophoresis was used in the final analysis. The number of detected spots from using this method allowed for calculation of bacterial diversity in complex soil systems [4].

Kottapallia et al (2008) used two-dimensional gel electrophoresis to create profiles of total seed proteins found in four separate peanut cultivars. The final analysis of their research identified 14 non-redundant proteins and differences in protein abundance between the cultivars. This is of significance regarding protein markers which may serve as a method of cultivar differentiation [5].

[1] Alberts et al. Molecular Biology of the Cell. 5th edition. New York, NY: Garland Science, 2002. 521-522. [2] Issaq HJ, Veenstra TD (2008) Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): advances and perspectives. Biotechniques 44: 697-700. [3] Chen T, Qi J, Xu J, Chen P, Tao A, Chen F, Chen W (2011) Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins. Agricultural Sciences in China 10: 1842-1850. [4] Liu G, Amemia T, Itoh K (2008) Two-Dimensional DNA Gel Electrophoresis Mapping: a Novel Approach to Diversity Analysis of Bacterial Communities in Environmental Soil. Journal of Bioscience and Bioengineering 105: 127-133. [5] Kottapallia KR, Payton P, Rakwal R, Agrawal GK, Shibato J, Burow M, Puppalaf N (2008) Proteomics analysis of mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritional, and allergenic proteins. Plant Science 175: 321-329.
 * References**