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High-Resolution Imaging of Cellular Proteins

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Cover of 'High-Resolution Imaging of Cellular Proteins'

Table of Contents

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    Book Overview
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    Chapter 1 Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.
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    Chapter 2 Antibody Production with Synthetic Peptides.
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    Chapter 3 High-Resolution Imaging of Cellular Proteins
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    Chapter 4 Preparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab')2, and Streptavidin.
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    Chapter 5 Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications.
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    Chapter 6 Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.
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    Chapter 7 Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.
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    Chapter 8 Using FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses.
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    Chapter 9 Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.
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    Chapter 10 Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging.
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    Chapter 11 Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins.
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    Chapter 12 Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.
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    Chapter 13 Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells.
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    Chapter 14 Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.
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    Chapter 15 High-Resolution Imaging of Cellular Proteins
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    Chapter 16 Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes.
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    Chapter 17 Pre-embedding Nanogold Silver and Gold Intensification.
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    Chapter 18 Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval.
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    Chapter 19 Pre- and Post-embedding Immunogold Labeling of Tissue Sections.
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    Chapter 20 High-Resolution Imaging of Cellular Proteins
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    Chapter 21 Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.
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    Chapter 22 High-Resolution Imaging of Cellular Proteins
Attention for Chapter 1: Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.
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Chapter title
Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.
Chapter number 1
Book title
High-Resolution Imaging of Cellular Proteins
Published in
Methods in molecular biology, January 2016
DOI 10.1007/978-1-4939-6352-2_1
Pubmed ID
Book ISBNs
978-1-4939-6350-8, 978-1-4939-6352-2
Authors

Jay M. Bhatt, Melanie L. Styers, Elizabeth Sztul Ph.D., Elizabeth Sztul, Bhatt, Jay M., Styers, Melanie L., Sztul, Elizabeth

Editors

Steven D. Schwartzbach, Omar Skalli, Thomas Schikorski

Abstract

Before the advent of molecular methods to tag proteins, visualization of proteins within cells required the use of antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Epitope tagging allows the detection of all proteins with existing sequence information, irrespective of the availability of antibodies directed against them. This technique involves the generation of DNA constructs that express the protein of interest tagged with an epitope that can be recognized by a commercially available antibody. Proteins can be tagged with a wide variety of epitopes using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into mammalian cell lines and, in most cases, tightly mimic the behavior of the endogenous protein. Tagged proteins exogenously expressed in cells provide different types of information depending on the subsequent detection approaches. Using immunofluorescence and immunoelectron microscopy with anti-tag antibodies, relative to known markers of cellular organelles, can provide information on the subcellular localization of the tagged protein and may provide clues regarding the protein's function. Immunofluorescence with anti-tag antibodies can also be utilized to assess the tagged protein's responses to cellular signals and pharmacological treatments. Immunoprecipitations with anti-tag antibodies can recover protein complexes containing the protein of interest, resulting in the identification of interacting proteins. Recovery of tagged proteins on affinity matrices allows their purification for use in biochemical assays. In addition, specialized fluorescent tags, such as the green fluorescent protein (GFP) allow the analysis of cellular dynamics in live cells in real time.

Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 12 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 12 100%

Demographic breakdown

Readers by professional status Count As %
Student > Master 5 42%
Student > Bachelor 4 33%
Researcher 2 17%
Unknown 1 8%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 6 50%
Agricultural and Biological Sciences 3 25%
Chemistry 1 8%
Unknown 2 17%