At present two-dimensional polyacrylamide gel electrophoresis (2-DE) is the most 
widely used proteomic tool, which enables simultaneous separation of even 
thousands of proteins with a high degree of resolution. The quality of 2-DE 
separation depends on the type of biological material used as a protein source. 
The presence of interfering compounds (e.g., phenols, as it is the fact in plant 
material including oat seeds) impedes 2-DE run. With the use of this technique 
it is possible to analyze the complex protein mixtures, characteristic protein 
fractions, as well as individual proteins.The purpose of this chapter is to 
describe the 2-DE technique (the separate stages of the first and the second 
dimension) for determining the oat protein composition (oat seed proteome), 
separation and preliminary identification of oat prolamin fractions. 
Electrophoretically separated proteins are identified on the basis of pI markers 
(identifying the location of both ends of an IPG strip) and on 2D SDS-PAGE 
standards. The gel images of oat proteins are analyzed with the help of 
ImageMaster 2D Platinum 6.0 program (Amersham Bioscience, part of GE Healthcare, 
Uppsala, Sweden). It allows finding unique spot identifiers for the occurrence 
of oat prolamin fractions in oat total proteins. The characteristic spots of 
similar shape and intensity (anchoring spots) and characteristic groups of spots 
can be searched for the purpose of identification.