Abstract
Two-dimensional (2D) gel electrophoresis combines isoelectric focusing in the first and SDS polyacrylamide gel electrophoresis in the second dimension to separate complex mixtures of proteins with unequalled resolution and sensitivity. It is well suited for the analysis of posttranslational protein modifications as most of them affect the isoelectric point and, therefore, the focusing behavior of the protein in the first dimension. It is particularly useful for low-abundance proteins, as it provides a first indication of PTMs, before establishing methods for protein isolation. For targeted proteomics of more abundant proteins, 2D electrophoresis itself may be the method of choice for the isolation of posttranslationally modified isoforms of the protein of interest for mass spectrometric analyses. Protein phosphorylation can be detected by use of phospho-specific stains or antibodies, or by comparing spot patterns of a protein sample before and after phosphatase treatment. Here we describe a simple method, combining 2D gel electrophoresis and western blot analysis with dephosphorylation by λ-phosphatase to analyze the phosphorylation status of oxophytodienoic acid reductase 3 in protein extracts from different organs of tomato and Arabidopsis plants.
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Acknowledgement
Funding from the German Research Foundation (DFG grant SCHA 591/6-1) is gratefully acknowledged. We also thank Bianca Pflüger for excellent technical assistance.
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Mayer, K., Albrecht, S., Schaller, A. (2015). Targeted Analysis of Protein Phosphorylation by 2D Electrophoresis. In: Schulze, W. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 1306. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2648-0_13
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DOI: https://doi.org/10.1007/978-1-4939-2648-0_13
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2647-3
Online ISBN: 978-1-4939-2648-0
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