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Two-dimensional gel electrophoresis (2-DE) offers the opportunity of separating several hundred proteins from a total yeast cellular extract. A detailed description is provided here of the different steps required for the separation and visualization of radiolabeled yeast proteins on high-resolution (24 cm x 20 cm) 2-D gels. Two methods of protein separation are described. They essentially differ by the way proteins are separated in the first dimension. One is based on the use of isoelectric focusing (IEF) gels (carrier ampholytes) and the other on the use of ready-made IPG gels (immobilines). These methods allow separating soluble proteins from a total yeast cellular extract with an isoelectric point ranging between pH 4.0 and 7.0 and a molecular weight ranging between 15,000 and 150,000.

We describe a novel methodology for the comparison of protein samples called differential gel exposure (DifExpo). This method is based on the coelectrophoresis on a two-dimensional (2-D) gel of two protein samples. The samples are differentiated from each other by in vivo radiolabelling, using (14)C- and (3)H-isotopes. After 2-D separation and transfer on a polyvinylidene difluoride membrane, the (3)H/(14)C ratio of each protein spot is determined by exposure to two types of imaging plates, one sensitive to (14)C and the other to both (14)C and (3)H. We showed that DifExpo allows us to compare the cellular levels of several hundred proteins of the yeast proteome. Its sensitivity is comparable to silver staining. We also showed that it can be used to investigate changes in the rate of synthesis of individual proteins.