Biology Reference
In-Depth Information
Chapter 23
DIGE Analysis of Plant Tissue Proteomes
Using a Phenolic Protein Extraction Method
Christina Rode , Traud Winkelmann , Hans-Peter Braun ,
and Frank Colditz
Abstract
Two-dimensional difference gel electrophoresis is an invaluable technique for the analysis of plant
proteomes. However, preparation of protein fractions from plant tissues is challenging due to the special
features of plant cells: a robust cell wall, large vacuoles which often contain high concentrations of organic
acids and a broad range of secondary metabolites like phenolic compounds and pigments. Therefore,
protein preparation for difference gel electrophoresis (DIGE) analyses has to be adapted. Here, we describe
both a phenolic protein extraction method for plant tissues and an adapted protocol for DIGE labeling
of the generated fractions.
Key words: Difference gel electrophoresis, Phenol extraction, Plant cell disruption, Plant proteomics,
Protein extraction
1. Introduction
Two-dimensional difference gel electrophoresis (2D DIGE)
technology allows separation of two differentially labeled protein
samples on one gel, thus eliminating gel-to-gel variability. A major
advantage of this technique is the possibility to identify even
very small or marginal differences in protein migration patterns
of two different fractions (occurring at the p I or Mr level) which
are hardly detectable by 2D gel electrophoresis on the basis of sep-
arate gels ( 1 ). Furthermore, the fl uorophores used in differential
protein labeling not only are very sensitive but also allow precise pro-
tein quantifi cation. In addition, this labeling allows a very sensitive
detection of proteins on gels.
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