Methods

Two-dimensional gel electrophoresis (2D-PAGE) of biological samples involves the separation of proteins based on two physico-chemical properties of proteins, i.e., isoelectric point (PI) and size (20). The first step is IEF, in which the proteins are focused according to their pi. The second step is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the proteins are further separated based on their migration rate. This step provides the two-dimensional map or total protein profile of a given sample. Comparisons of such profiles, or maps, help in determination of differential protein expression. This technique also allows for the screening thousands of proteins at once and thereby providing information about the post-translational modifications, which result in changes in total protein charge (i.e., shift in the position of the protein spot on the gel). In our laboratory, we invoked the use of a parallel analysis in which the 2D gel map is coupled with 2D immunochemical detection of protein carbonyls derivatized by 2,4-dinitrophenylhyrdazine followed by mass spectrometry analysis to identify proteins of particular interest (8, 11, 12, 15, 16, 18, 21). The redox proteomics method used in our laboratory is outlined in Fig. 11.1.

3.1. Sample Derivatization for the Detection of Protein Carbonyls

1. Prepare 10% (w/v) tissue or cell homogenate in smaple homogenization buffer (10 g of tissue per 100 ml of sample homogenization buffer). Take a small amount of homogenized tissue and sonicate for 10 s on ice.

Sample (Protein mixture)

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