2-DE Separation and Identification of Oat (Avena sativa L.) Proteins and Their Prolamin Fractions.

Abstract

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.