Protocol

Plant Proteomics

Volume 355 of the series Methods in Molecular Biology pp 157-174

Two-Dimensional Differential In-Gel Electrophoresis (DIGE) of Leaf and Roots of Lycopersicon esculentum

  • Matthew KeelerAffiliated withDepartment of Biochemistry and Molecular Biophysics, The University of Arizona
  • , Jessica LetarteAffiliated withBio5 Institute for Collaborative Bioresearch, The University of Arizona
  • , Emily HattrupAffiliated withDepartment of Biochemistry and Molecular Biophysics, The University of Arizona
  • , Fatimah HickmanAffiliated withBio5 Institute for Collaborative Bioresearch, The University of Arizona
  • , Paul A. HaynesAffiliated withBio5 Institute for Collaborative Bioresearch, The University of Arizona

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Abstract

In this report we present a detailed protocol for the analysis of differential protein expression between two plant tissue samples. The protocol involves harvesting of leaves and roots from mature tomato plants, preparing protein extracts from the harvested tissues, fluorescent labeling of each sample prior to differential in-gel electrophoresis (DIGE), first- and second-dimension electrophoretic separations, and image analysis to visualize and quantify differential protein expression. This protocol is adaptable for use with a wide variety of plant materials and can be used to measure protein expression changes occurring in response to abiotic stress, biotic stress, genetic manipulation, selective breeding, and many other conditions. In addition to the detailed protocol, we also present the results of a representative experiment analyzing subtle changes in protein expression in the roots of tomato plants grown under control and salt-stress conditions.

Key Words

2D protein gel electrophoresis differential in-gel electrophoresis DIGE plant protein tomato CyDye protein staining differential wavelength fluorescence imaging Lycopersicon esculentum