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Proteomic Approaches in Medical and Clinical Research:
Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis (2DGE) is used to separate and quantitate complex protein mixtures. Typically, cell lysates are fractionated in some manner, the fractions are desalted and exchanged into a suitable buffer, and the protein concentration is determined. Proteins are separated by pI using isoelectrofocusing (the first dimension) then isoelectric strips are placed onto an SDS-PAGE gel and the proteins are separated by size (second dimension). The complexity of the mixture and the nature of the sample will dictate the pH range of the isoelectric strip to use and the size of the gel.
Usually gels representing control and treated samples are compared. Analytical software is used to match corresponding spots in the gels and compute relative differences in their intensities, providing a quantitative picture of global protein expression changes within the sample. Spots with significant differences are typically excised and the proteins digested into peptides using an endopeptidase such as trypsin. The peptides are extracted from the gel spots and then analyzed using mass spectrometry.
Alternatively, proteins in different samples can be differentially labeled then pooled and run on the same gel. The dyes on the labels are separately imaged and quantitative differences between the labeled proteins are calculated. The CNRU Mass Spectrometry Core Facility is also equipped for these Difference In Gel Electrophoresis (DIGE) experiments.
Please follow the links to obtain more information on sample fractionation, sample preparation and formats for first dimension and second dimension separations.
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