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Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy

Histochemical methods allow for identification and localization of various components within the tissue. Such information on the spatial heterogeneity is not available with biochemical methods. However, there is limitation of the specificity of such detection in context of com ...

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Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery

The budding yeast (Saccharomyces cerevisiae) can serve as a unique experimental system for functional studies of heterologous genes, allowing not only complementation of readily available yeast mutations but also generation of overexpression phenotypes and in some cases also r ...

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Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues

Plant cell walls are structurally diverse macromolecular composites. One of our best methodologies to determine the temporal and spatial regulation of cell wall polysaccharides in relation to development are monoclonal antibody (MAB) and carbohydrate-binding module (CBM) pr ...

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Image Analysis: Basic Procedures for Description of Plant Structures

This chapter gives examples of basic procedures of quantification of plant structures with the use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a wid ...

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Automated Microscopy in Forward Genetic Screening of Arabidopsis

Tightly controlled spatiotemporal specificity of gene expression is intrinsic to developmental and adaptation responses of living systems throughout the kingdoms. Forward genetic screens employing well-characterized reporter lines can be used to identify as yet unknown g ...

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Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells

Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions t ...

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Visualizing and Quantifying the In Vivo Structure and Dynamics of the Arabidopsis Cortical Cytoskeleton Using CLSM and VAEM

The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo stud ...

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Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces

Sequential replica method facilitates in vivo imaging of plant surface and provides data sufficient for detailed computation of geometry and growth. It enables obtaining a series of high-resolution images visualizing details of the examined surface. Series of molds, made in dental po ...

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Time-Lapse Imaging of Developing Meristems Using Confocal Laser Scanning Microscope

Analysis of shoot meristem shape and gene expression pattern has been conducted in many species over the past decades. Recent live imaging techniques have allowed an unprecedented accumulation of data on the biology of meristematic cells, as well as a better understanding of the molecular a ...

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The Minimal Information About a Proteomics Experiment (MIAPE) from the Proteomics Standards Initiative

During the last 10 years, the Proteomics Standards Initiative from the Human Proteome Organization (HUPO-PSI) has worked on defining standards for proteomics data representation as well as guidelines that state the minimum information that should be included when reporting a prote ...

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Precipitation of Champagne Base Wine Proteins Prior to 2D Electrophoresis

Numerous methods have been employed to depict the protein content of wines. Among them, two-dimensional electrophoresis (2D-E) presents a powerful resolution, but has been poorly applied to wine. Furthermore, 2D-E was coupled with various extraction methods of proteins without any re ...

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Beer and Wort Proteomics

Proteome analysis provides a way to identify proteins related to the quality traits of beer. A number of protein species in beer and wort have been identified by two-dimensional gel electrophoresis combined with enzyme digestion such as trypsin, followed by mass spectrometry analyses and ...

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Immunoproteomics Analysis of Food Allergens

Immunoblot-coupled proteomics based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), namely, immunoproteomics, has been used for comprehensive identification of food allergens, because it is a simple and inexpensive tool for rapid identification of sev ...

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Proteotyping of Holm Oak (Quercus ilex subsp. ballota) Provenances Through Proteomic Analysis of Acorn Flour

Proteomics has become a powerful tool to characterize biodiversity and natural variability in plant species, as well as to catalogue and establish phylogenetic relationships and distances among populations, provenances or ecotypes. In this chapter, we describe the standard prot ...

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Class III Peroxidases

Class III peroxidases are heme-containing proteins of the secretory pathway with an extremely high number of isoenzymes, indicating the tremendous and important functions of this protein family. This chapter describes fractionation of the cell in subproteomes, their separation ...

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Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulf ...

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Separation of Membrane Protein Complexes by Native LDS-PAGE

Gel electrophoresis has become one of the most important methods for the analysis of proteins and protein complexes in a molecular weight range of 1–107 kDa. The separation of membrane protein complexes remained challenging to standardize until the demonstration of Blue Native PAGE in 1991 ...

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Assay for Proteasome-Dependent Protein Degradation and Ubiquitinated Proteins

The ubiquitin-26S proteasome system (UPS) plays a crucial role in selective removal of short-lived target proteins, archiving fine-tuning of post-translation levels of the target proteins. Recently a number of ubiquitin ligases (E3) have been reported as essential regulators of var ...

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Rapid and High-Throughput N-Glycomic Analysis of Plant Glycoproteins

Glycoprotein is a major element in higher organisms including mammalians and plants. It is widely accepted that variation in cellular N-glycome is related to modulation in dynamic cellular mechanisms such as cell-cell adhesion, cell activation, and malignant alterations in mammal ...

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N-Glycoprotein Enrichment by Lectin Affinity Chromatography

Lectins are proteins that bind to sugars with varying specificities and several have been identified that show differential binding to structurally variable glycans attached to glycoproteins. Consequently, lectin affinity chromatography represents a valuable tool for gly ...

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