Two-Dimensional Gel Electrophoresis of Total Yeast Proteins

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 � 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.