Two-Dimensional Electrophoresis
The classical method of analyzing differential expressed proteins between two samples was developed 30 years ago (O'Farrell PH, (1975) High Resolution Two-Dimensional Electrophoresis of Proteins. JBC 250 (10) 400-4021). The method is based on separation of a protein mixture based on two independent physical properties of proteins, the protein's pI and molecular weight. Although the method is tedious and archaic compared to the current MS based analysis of differential expressed protein it provides a visual representations of the proteins in the cell. Two-Dimensional Electrophoresis has been improved over the years allowing reproducibility of results and increased sensitivity. One improvement of two-dimensional electrophoresis is the immobilized pH gradient (IPG), which allows reproducibility. Two-dimensional electrophoresis also allows the detection of less abundant protein, which is especially important when a few proteins make up the majority of a complex protein mixture. The chose of pH gradients also can improve resolution of proteins as well as increase the amount of low abundance proteins with increased loading capacity. The combination of various detection methods with two-dimensional electrophoresis, ie, selective staining, in vitro radio-labeling or western blot techniques allows the detection of post translational modifications that may not be detectable by direct MS techniques because of the low abundance of the PTM on the entire population of a single protein. The Proteomic Analysis Core can assist in all facets of proteomic studies from the preparation of samples to the preparation of the sample for MS analysis. Proteomic Analysis of a sample with Two-Dimensional Electrophoresis consists of the:
Experimental Design
Sample Preparation (link to 2D sample prep)
Sample Resolution
IEF
SDS-PAGE
Sample Imaging (link to typhoon))
Differential Expression Evaluation
Spot Picking (link to protepic))
Sample preparation for MS analysis (link to prprep)
Digestion
Extraction
Spotting on MALDI target or preparation for electrospray
Protein Identification via MS
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