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The aim of Plant Proteomics: Methods and Protocols is to present up-- date methods and protocols used by recognized scientists in the world of plant proteomics. If this world was a very small one twenty-five years ago when the first papers were published, it has since experienced exponential growth, and in most countries around the world there are laboratories working on plant proteomics. Two-dimensional gel electrophoresis is still the basic method used, but it has been improved greatly with IPG in the first dimension (Chapter 13) and with new detection methods with fluorochromes (Chapters 14 and 15). Signi- cant progress has been achieved in protein extraction, which is particularly difficult with plant tissues containing phenols, proteases, and other secondary metabolites that interfere with proteins. Standard procedures have been op- mized (Chapters 1 and 2) for peculiar tissues (Chapters 3, 4, and 5) and cellular compartments (Chapters 6 to 10). These methods rely on improvements made in the solubilization of proteins from membranes (Chapters 11 and 12). Mass spectrometry was a revolution that permitted the high throughput identifi- tion of proteins separated by 2D gels (Chapters 19 and 20) but also from blue native 1D gels (Chapters 27 and 28) despite the fact that Edman sequencing can still be useful (Chapter 18). Associated with other techniques such as 2DLC or LC of intact proteins, mass spectrometry also permits the identification of polypeptides from complexes (Chapters 21 and 22).
Sommario
Total Protein Extraction with TCA-Acetone.- Phenol Extraction of Proteins for Proteomic Studies of Recalcitrant Plant Tissues.- Protein Extraction from Cereal Seeds.- Protein Extraction from Xylem and Phloem Sap.- Protein Extraction from Woody Plants.- Isolation of Chloroplast Proteins from Arabidopsis thaliana for Proteome Analysis.- Isolation and Subfractionation of Plant Mitochondria for Proteomic Analysis.- Extraction of Nuclear Proteins from Root Meristematic Cells.- Extraction of Nuclear Proteins.- Isolation of Cell Wall Proteins from Medicago sativa Stems.- Plant Plasma Membrane Protein Extraction and Solubilization for Proteomic Analysis.- Detergents and Chaotropes for Protein Solubilization before Two-Dimensional Electrophoresis.- Two-Dimensional Electrophoresis for Plant Proteomics.- Visible and Fluorescent Staining of Two-Dimensional Gels.- Two-Dimensional Differential In-Gel Electrophoresis (DIGE) of Leaf and Roots of Lycopersicon esculentum.- Quantitative Analysis of 2D Gels.- Multivariate Data Analysis of Proteome Data.- Edman Sequencing of Proteins from 2D Gels.- Peptide Mass Fingerprinting.- Protein Identification Using Nano Liquid Chromatography-Tandem Mass Spectrometry.- Two-Dimensional Nanoflow Liquid Chromatography-Tandem Mass Spectrometry of Proteins Extracted from Rice Leaves and Roots.- Separation, Identification, and Profiling of Membrane Proteins by GFC/IEC/SDS-PAGE and MALDI TOF MS.- The PROTICdb Database for 2-DE Proteomics.- Identification of Phosphorylated Proteins.- Plant Proteomics and Glycosylation.- Blue-Native Gel Electrophoresis for the Characterization of Protein Complexes in Plants.- Electroelution of Intact Proteins from SDS-PAGE Gels and Their Subsequent MALDI-TOF MS Analysis.- Generation of Plant Protein Microarrays and Investigation of Antigen-Antibody Interactions.- Phosphorylation Studies Using Plant Protein Microarrays.
Riassunto
The aim of Plant Proteomics: Methods and Protocols is to present up-- date methods and protocols used by recognized scientists in the world of plant proteomics. If this world was a very small one twenty-five years ago when the first papers were published, it has since experienced exponential growth, and in most countries around the world there are laboratories working on plant proteomics. Two-dimensional gel electrophoresis is still the basic method used, but it has been improved greatly with IPG in the first dimension (Chapter 13) and with new detection methods with fluorochromes (Chapters 14 and 15). Signi- cant progress has been achieved in protein extraction, which is particularly difficult with plant tissues containing phenols, proteases, and other secondary metabolites that interfere with proteins. Standard procedures have been op- mized (Chapters 1 and 2) for peculiar tissues (Chapters 3, 4, and 5) and cellular compartments (Chapters 6 to 10). These methods rely on improvements made in the solubilization of proteins from membranes (Chapters 11 and 12). Mass spectrometry was a revolution that permitted the high throughput identifi- tion of proteins separated by 2D gels (Chapters 19 and 20) but also from blue native 1D gels (Chapters 27 and 28) despite the fact that Edman sequencing can still be useful (Chapter 18). Associated with other techniques such as 2DLC or LC of intact proteins, mass spectrometry also permits the identification of polypeptides from complexes (Chapters 21 and 22).
