Ian Ashley

2-D electrophoresis and analysis
Basic Description:

This is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. The 2-D process begins with sample preparation. Proper sample preparation is absolutely essential for a good 2-D result. This technique is used for proteomics analysis, differentiation of cells, detection of diseases and cancer research.



Purpose of Technique:

This technique sorts proteins according to two independent properties in two discrete steps: the first-dimension step uses isoelectric focusing (IEF), to separate proteins according to their isoelectric points. The second-dimension step uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE), to separate proteins according to their molecular weights. After these two steps have been performed, the resulting two-dimensional array of spots are visualized and analyzed.


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Figure 1 is the 2D gels (stained)

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Figure 2 is the instrument modern form

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Fig. 3: Process of 2D-electrophoresis


Origin and History:

2-DE was first independently introduced by O'Farrell and Klose in 1975. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins.

Recent research:

This article talks about the BRCA2 pathways in breast cancer. The instrument is used to detect the protein, but the problem is that the gene is bigger than an average gene. 2DE is used to quantify the proteins and analyses the size and PI points. The researchers can know if the gene affects the pathways of breast cancer or not. The gene is harmful correspond with a fivefold increase in a woman’s risk for breast cancer, from about 12% without the mutations to 60% with them (Bennett, 2013).

Analysis of 2D-GE images is difficult because, current software has proven to be not very effective and usually requires manual adjustment and refinement of the computer generated images. The authors of this article developed an effective new technique for the detection and deconstruction of over-saturated protein spots (Natale et al., 2012).

The goal of this study was to add to the knowledge of the Perkinsus spp. proteome. The authors extracted proteins from three species of the genus Perkinsus and used 2D-electrophoresis to analyze and compare them. They were able to distinguish 2000 protein spots from each of the three species and determine that two of the species were closer to each other than they were compared to the other species (Fernandez-Boo et al., 2014).

In this article, 2D-electrophoresis was used to evaluate decellularization effectiveness and to identify potentially bioactive protein remnants in a human vocal fold mucosa model. The use of the 2D-E provided the dual benefits of extensive sample fractionation prior to LC-MS/MS and parallel immunoprofiling of system-wide phosphorylation and glycosylation (Welham et al., 2013).

References:

Bennett, J. (2013). Revealing BRCA2 Pathways in Cancer with Bio-Rad's V3 Western Workflow. Bioradiations. Retrieved October 7, 2014, from http://www.bioradiations.com/focus-on-applications/57-2-d-electrophoresis-and-analysis/1410-revealing-brca2-pathways-in-cancer-with-the-v3-western-workflow

Natale, M., Caiazzo, A., Bucci, E.M., & Ficarra, E. (2012). A Novel Gaussian Extrapolation Approach for 2D Gel Electrophoresis Saturated Protein Spots. Genomics, Proteomics & Bioinformatics, 10(6), 336-344. doi: 10.1016/j.gpb.2012.06.005

Fernandez-Boo, S., Chicano-Galvez, E., Alhama, J., Barea, J.L., Villalba, A., & Cao, A. (2014). Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry. Journal of Invertebrate Pathology, 118, 47-58. doi: 10.1016/j.jip.2014.02.011

Welham, N.V., Chang, Z., Smith, L.M., & Frey, B.L. (2013). Proteomic analysis of a decellularized human vocal fold mucosa scaffold using 2D electrophoresis and high-resolution mass spectrometry. Biomaterials, 34(3), 669-676. doi: 10.1016/j.biomaterials.2012.09.050

Fig. 3 image. Proteomics and Its use in Obtaining Superior Soybean Genotypes. Retrieved online October 11, 2014. <www.intechopen.com/source/html/38177/media/image4.png>