ABSTRACT
This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.
Subject(s)
Electrophoresis, Gel, Two-Dimensional , Isoelectric Focusing , Plant Proteins , Electrophoresis, Gel, Two-Dimensional/methods , Plant Proteins/isolation & purification , Plant Proteins/analysis , Isoelectric Focusing/methods , Proteomics/methods , Plants/chemistry , Mass Spectrometry/methodsABSTRACT
Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be extracted from plant tissues, which are rather complex than other types of biological material. Different protocols have been applied depending on the protein source, such as seeds, pollen, leaves, roots, and flowers. Total protein amounts must also be determined before conducting gel electrophoresis. The most common methodologies include PAGE under native or denaturing conditions. Both procedures are used consequently for protein identification and characterization via mass spectrometry. Additionally, various staining procedures are available to visualize protein bands in the gel, facilitating the software-based digital evaluation of the gel through image acquisition.