|
|
Last Updated September, 2002
Foreword by Kai Simons
Preface
Contributors
1 Cell Culture
2 Preparation and Isolation of Cells
3 Subcellular Fractionation and Isolation of Organelles
4 Microscopy
4A Organelle Atlas
5 Characterization of Cellular Proteins
6 Electrophoresis and Immunoblotting
7 Protein Labeling and Immunoprecipitation
8 Cell Cycle Analysis
9 Cell Adhesion
10 Extracellular Matrix
11 In Vitro Reconstitution
12 Cell Motility
13 Organelle Motility
14 Signal Transduction: Protein Phosphorylation
15 Protein Trafficking
16 Antibodies as Cell Biological Tools
17 Macromolecular Interaction in Cells
18 Cellular Aging and Death
19 Whole Organism and Tissue Analysis
Appendices
Index
--------------------------------------------------------------------------------
1 Cell Culture
1.1 Basic Techniques for Mammalian Cell Tissue Culture
1.2 Media for Culture of Mammalian Cells
1.3 Aseptic Technique for Cell Culture
1.4 Sterilization and Filtration
1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
1.6 Media and Culture of Yeast
2 Preparation and Isolation of Cells
2.1 Establishment of Fibroblast Cultures
2.2 Preparation and Culture of Human Lymphocytes
2.3 Preparation of Endothelial Cells
2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
2.5 Laser Capture Microdissection
3 Subcellular Fractionation and Isolation of Organelles
3.1 Overview of Cell Fractionation
3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density
Gradient Centrifugation
3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density
Gradient Centrifugation
3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces
cerevisiae
3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and
Density Gradient Centrifugation
3.10 Isolation of Nuclei and Nuclear Membranes from Animal Tissues
3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
4 Microscopy
4.1 Proper Alignment and Adjustment of the Light Microscopes
4.2 Fluorescence Microscopy
4.3 Immunofluorescence Staining
4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
4.5 Basic Confocal Microscopy
4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells
and Tissues
4.7 Cryo-Immunogold Electron Microscopy
4.8 Correlative Video Light/Electron Microscopy
4.9 Polarization Microscopy
4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living
Cells
4A Organelle Atlas
This atlas contains images of cellular organelles visualized
using a wide variety of reagents and techniques and demonstrates the diversity
of methods for exploring the cell.
5 Characterization of Cellular Proteins
5.1 Overview of Physical State of Proteins Within Cells
5.2 Determining the Topology of an Integral Membrane Protein
5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose
Density Gradients
5.4 Analysis of the Association of Proteins with Membranes
5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
6 Electrophoresis and Immunoblotting
6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
6.2 Immunoblotting and Immunodetection
6.3 Detection and Quantitation of Radiolabeled Proteins in Gels
6.4 Two-Dimensional Gel Electrophoresis
6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
6.6 Staining Proteins in Gels
6.7 Agarose Gel Electrophoresis of Proteins (New, July 2002)
6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots (New,
October 2002)
6.9 Digital Electrophoresis Analysis (New, October 2002)
7 Protein Labeling and Immunoprecipitation
7.1 Metabolic Labeling with Amino Acids
7.2 Immunoprecipitation
7.3 Metabolic Labeling with Sulfate
7.4 Metabolic Labeling with Fatty Acids
7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
7.7 Metabolic Labeling and Immunoprecipiation of Drosophila Proteins
7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
7.9 Analysis of Oxidative Modification of Proteins
7.10 Radioiodination of Cellular Proteins (New, July 2002)
8 Cell Cycle Analysis
8.1 Overview of the Cell Cycle
8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
8.4 Determining Cell Cycle Stages by Flow Cytometry
8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
9 Cell Adhesion
9.1 Cell-Substrate Adhesion Assays
9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
9.3 Cadherin-Dependent Cell-Cell Adhesion
9.4 Analyzing Integrin-Dependent Adhesion (Revised, July 2002)
9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
10 Extracellular Matrix
10.1 Overview of Extracellular Matrix
10.2 Preparation of Basement Membrane Components from EHS Tumors
10.3 Preparation of Gelled Substrates
10.4 Preparation of Extracellular Matrices Produced by Cultured Cells
10.5 Purification of Fibronectin
10.6 Purification of Vitronectin
10.7 Proteoglycan Isolation and Analysis
10.8 Matrix Metalloproteinases
10.9 Preparation of Extracellular Matrices Produced by Cultured Fibroblasts
(New, October 2002)
11 In Vitro Reconstitution
11.1 Overview of Eukaryotic In Vitro Translation and Expression
Systems
11.2 In Vitro Translation
11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian
Cells
11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
11.5 In Vitro Analysis of SV40 DNA Replication
11.6 In Vitro Transcription
11.7 Nuclear Import in Digitonin-Permeabilized Cells
11.8 In Vitro Translation Using HeLa Extract
11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
11.10 Preparation and Use of Interphase Xenopus Egg Extracts
11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
11.13 Mitotic Spindle Assembly In Vitro
11.14 Analysis of RNA Export Using Xenopus Oocytes
11.15 In Vitro Analysis of Peroxisomal Protein Import
11.16 In Vitro Analysis of Chloroplast Protein Import
12 Cell Motility
12.1 Chemotaxis Assays for Eukaryotic Cells
12.2 Invasion Assays
12.3 Cell Traction
12.4 Cell Wounds Assays
12.5 Dictyostelium Cell Dynamics
13 Organelle Motility
13.1 Microtubule-Organelle Motility Assays
13.2 In Vitro Assay to Study Translocation of Actin by Myosin
13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
13.4 Movement of Nuclei
14 Signal Transduction: Protein Phosphorylation
14.1 Overview of Protein Phosphorylation
14.2 Immunological Detection of Phosphorylation
14.3 Detection of MAP Kinase Signaling
14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
14.5 Phosphoamino Acid Analysis
15 Protein Trafficking
15.1 Overview of Protein Trafficking in the Secretory and Endocytic
Pathways
15.2 Use of Glycosidases to Study Protein Trafficking
15.3 Endocytosis: Biochemical Analyses
15.4 Determining Protein Transport to the Plasma Membrane
15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
16 Antibodies as Cell Biological Tools
16.1 Production of Monoclonal Antibodies
16.2 Production of Polyclonal Antisera
16.3 Purification of Immunoglobulin G
16.4 Fragmentation of Immunoglobulin G
16.5 Antibody Conjugates for Cell Biology
16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated
Peptides
17 Macromolecular Interaction in Cells
17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance
Energy Transfer (FRET) Microscopy
17.2 Identification of Protein Interactions by Far Western Analysis
17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
18 Cellular Aging and Death
18.1 Current Concepts in Cell Death
18.2 Analysis of Caspase Activation During Apoptosis
18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological
Criteria )
18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
19 Whole Organism and Tissue Analyses
19.1 Overview of Metastasis Assays
19.2 Tail Vein Assay of Cancer Metastasis
19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis
of Gene Expression After High-Fidelity T7-Based RNA Amplification (New, October
2002)
19.4 SAGE Analysis from 1 mg of Total RNA (New, October 2002)
Appendices
A.1 USEFUL INFORMATION AND DATA
A. Useful Measurements and Data
B. Compendium of Drugs Commonly Used in Cell Biology
C. Identification of Motifs in Protein Sequences
D. Safe Use of Radioisotopes
E. Absorption and Emission Maxima of Common Fluorophores
F. Importing Biological Materials
G. Centrifuges and Rotors
A.2 LABORATORY STOCK SOLUTIONS AND EQUIPMENT
A. Common Stock Solutions, Buffers, and Media
B. Media Formulations
C. Standard Laboratory Equipment
A.3 COMMONLY USED TECHNIQUES
A. Molecular Biology Techniques
B. Spectrophotometric Determination of Protein Concentration (Revised, July
2002)
C. Dialysis and Concentration of Protein Solutions
D. Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
E. Silanizing Glassware
F. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
G. Micro RT-PCR
H. The Colorimetric Detection and Quantitation of Total Protein (New, July 2002)