Abstract

Our two-dimensional gel electrophoresis (2DE) protocol has been continuously improved in our laboratory since its inception 30 years ago. An updated version is presented here. This protocol is a result of our experience in proteome analysis of tissue extracts, cultured cells (mammalian, yeast, and bacteria), cellular organelles, and subcellular fractions. Many modifications and suggestions emerging in our lab as well as in the literature were tested and integrated into our improved protocol if helpful. Importantly we use (a) large (46 x 30 cm) gels to achieve a high resolution and (b) ready-made gel solutions produced in large batches and stored frozen, a prerequisite, among others, for our very high reproducibility. Employing the 2DE method described here we demonstrated that protein patterns separating more than 10,000 protein spots can be obtained from mouse tissue. This is the highest resolution reported in the literature for 2DE of complex protein mixtures so far. Our 2DE patterns are of high quality with regard to spot shape and intensity as well as background. The reproducibility of the protein patterns is shown to be extremely satisfactory. New staining methods such as differential in gel electrophoresis (DIGE) and the latest 2DE gel evaluation software are compatible to our 2DE protocol. Using suitable staining protocols proteins can easily be identified by mass spectrometry.