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基本説明
Providing condensed descriptions of more than 500 methods compiled from Current Protocols in Cell Biology.
Full Description
Providing condensed descriptions of more than 500 methods compiled from Current Protocols in Cell Biology, this text thoroughly explores cell biology in an easily accessible, hands-on format. Short Protocols in Cell Biology is an authoritative and indispensable guide for all life scientists and researchers who are looking to improve their understanding of cell biology methods. Key Features:* Designed to provide quick access to step-by-step instructions for the essential methods used in every major area of cell biological research* Contains methods from every aspect of cell biologyAC - everything needed to study the basic structure and functions of cells at both the molecular and cellular levels
Table of Contents
Preface xvii
Contributors xxi
1 Cell Culture
1.1 Basic Techniques for Mammalian Cell 2
Tissue Culture 1-
Basic Protocol: Trypsinizing and 3 (1)
Subculturing Cells from a Monolayer 1-
Alternate Protocol: Passaging Cells in 3 (1)
Suspension Culture 1-
Support Protocol 1: Freezing Human Cells 4 (1)
Grown in Monolayer Cultures 1-
Support Protocol 2: Freezing Cells Grown in 4 (1)
Suspension Culture 1-
Support Protocol 3: Thawing and Recovering 5 (1)
Human Cells 1-
Support Protocol 4: Determining Cell Number 5 (1)
and Viability with a Hemacytometer and
Trypan Blue Staining 1-
Support Protocol 5: Preparing Cells for 6
Transport 1-
1.2 Media for Culture of Mammalian Cells 1- 1 (11)
Basic Protocol 1: Preparation of 7 (1)
Serum-Containing Media 1-
Basic Protocol 2: Preparing Media for 8 (2)
Reduced-Serum or Serum-Free Growth 1-
Basic Protocol 3: Preparation of Selective 10 (1)
Media: HAT Medium 1-
Basic Protocol 4: Growth of Transformed 10 (1)
Cells in Soft Agar 1-
Support Protocol 1: pH Control in Media 1- 11 (1)
Support Protocol 2: Use of Antibiotics in 11 (1)
Media 1-
1.3 Aseptic Technique for Cell Culture 1. .12
Basic Protocol 1: Aseptic Technique 1- 12 (2)
Basic Protocol 2: Use of the Laminar-Flow 14 (1)
Hood 1-
1.4 Sterilization and Filtration 1- 15 (6)
Basic Protocol 1: Autoclaving Liquids 1- 15 (2)
Alternate Protocol: Autoclaving Dry Goods 1- 17 (1)
Basic Protocol 2: Dry-Heat Sterilization 17 (1)
and Depyrogenation 1-
Basic Protocol 3: Use of Disinfectants: 70% 18 (1)
Ethanol 1-
Filter Sterilization of Solutions 1- 19 (1)
Basic Protocol 4: Vacuum Filtration 1- 19 (1)
Basic Protocol 5: Small-Volume 20 (1)
Positive-Pressure Filtration of Nonaqueous
Solutions 1-
1.5 Assessing and Controlling Microbial 21 (5)
Contamination in Cell Cultures 1-
Basic Protocol 1: Testing for Bacterial and 21 (2)
Fungal Contaminants 1-
Basic Protocol 2: Testing for Mycoplasma 23 (2)
Contamination by Direct Culture 1-
Basic Protocol 3: Use of Antibiotics to 25 (1)
Control Microbial Contamination 1-
1.6 Media and Culture of Yeast 1- 26
Preparation of Media 1- 27 (5)
General Considerations for Culturing Yeast 32
1-
2 Preparation and Isolation of Cells
2.1 Establishment of Fibroblast Cultures 2- 2 (4)
Basic Protocol
2.2 Preparation and Culture of Human 6 (4)
Lymphocytes 2-
Basic Protocol 1: Preparation of 6 (2)
Lymphocytes by Ficoll-Hypaque Gradient
Centrifugation 2-
Basic Protocol 2: Preparation of 8 (1)
Monocytes/Macrophages and "Dendritic-Like"
Cells from Lymphocyte Populations 2-
Basic Protocol 3: Positive Selection of T 9 (1)
and B Cells by Monoclonal Antibody-Coated
Magnetic Beads 2-
2.3 Preparation of Endothelial Cells from 10 (2)
Human Umbilical Vein 2-
Basic Protocol 2- 10 (2)
2.4 Generation of Continuously Growing B Cell 12
Lincs by Epstein-Barr Virus Transformation 2-
Basic Protocol 2- 12
2.5 Laser Capture Microdissection 2- -13 (15)
Basic Protocol: Isolation of a Pure Cell 13 (3)
Population from Tissue Sections 2-
Support Protocol: Hematoxylin and Eosin 16
Staining of Tissues for LCM 2-
3 Subcellular Fractionation and Isolation of
Organelles
3.1 Overview of Cell Fractionation 3- 2 (8)
Basic Principles of Centrifugation 3- 3 (1)
Instrumentation 3- 4 (3)
Fractionation Media 3- 7 (2)
Evaluation of Fractionation 3- 9 (1)
Definitive Procedures 3- 10 (1)
3.2 Isolation of Rat Hepatocyte Plasma 10 (7)
Membrane Sheets and Plasma Membrane Domains 3-
Basic Protocol 1: Isolation of Plasma 10 (2)
Membrane Sheets 3-
Support Protocol 1: Assay for Alkaline 12 (2)
Phosphodiesterase I Activity 3-
Basic Protocol 2: Isolation of Plasma 14 (1)
Membrane Domains 3-
Support Protocol 2: Assay for K+-Stimulated 14 (1)
p-Nitrophenylphosphatase Activity 3-
Support Protocol 3: Assay for 15 (1)
5'-Nucleotidase Activity 3-
Support Protocol 4: Indirect 16 (1)
Immunofluorescent Detection of Proteins
Associated with Plasma Membrane Sheets 3-
3.3 Isolation of Golgi Membranes from Tissues 17 (7)
and Cells by Differential and Density
Gradient Centrifugation 3-
Basic Protocol 1: Rapid Isolation of Golgi 18 (1)
Membranes from Rat Liver Using a Sucrose
Density Barrier 3-
Basic Protocol 2: Isolation of Golgi 19 (2)
Membranes from a Rat Liver Light
Mitochondrial Fraction by Flotation Through
a Discontinuous Sucrose Gradient 3-
Basic Protocol 3: Isolation of Golgi 21 (1)
Membranes from Cultured Cells by Flotation
Through a Discontinuous Sucrose Gradient 3-
Basic Protocol 4: Isolation of Golgi 21 (2)
Membranes from a Microsomal Fraction of
Hepatocytes in a Self-Generated Gradient of
Iodixanol 3-
Support Protocol: Assay for UDP-Galactose 23 (1)
Galactosyltransferase 3-
3.4 Isolation of Lysosomes from Tissues and 24 (4)
Cells by Differential and Density Gradient
Centrifugation 3-
Basic Protocol 1: Isolation of Lysosomes 24 (2)
from Rat Liver Using a Self-Generated
Percoll Gradient 3-
Basic Protocol 2: Isolation of Lysosomes 26 (1)
from Human HL-60 Cultured Cells Using a
Self Generated Percoll Gradient 3-
Support Protocol 1: Assay for Acid 27 (1)
Phosphatase 3-
Support Protocol 2: Assay for ﾟ-1V 27 (1)
Acetylglucosaminidase 3-
3.5 Isolation of Mitochondria from Tissues 28 (5)
and Cells by Differential Centrifugation 3-
Basic Protocol 1: Preparation of the Heavy 28 (2)
Mitochondrial Fraction from Rat Liver 3-
Basic Protocol 2: Large-Scale Preparation 30 (1)
of Mitochondria from Bovine Heart 3-
Basic Protocol 3: Preparation of 31 (1)
Mitochondria from Skeletal Muscle 3-
Basic Protocol 4: Preparation of 32 (1)
Mitochondria from Cultured Cells 3-
Basic Protocol 5: Preparation of 32 (1)
Mitochondria from Yeast (Saccharomyces
cerevisiae) 3-
3.6 Purification of a Crude Mitochondrial 33 (6)
Fraction by Density-Gradient Centrifugation 3-
Basic Protocol 1: Resolution of a Rat Liver 34 (1)
Mitochondrial Fraction in a Continuous
Sucrose Gradient 3-
Basic Protocol 2: Isolation of Mitochondria 35 (1)
from Rat Brain Using a Discontinuous
Percoll Gradient 3-
Basic Protocol 3: Resolution of a 36 (1)
Mitochondrial Fraction in a Self-Generated
Percoll Gradient 3-
Support Protocol 1: Succinate Dehydrogenase 37 (1)
Assay for Mitochondria 3-
Support Protocol 2: 3-Galactosidase Assay 37 (1)
for Lysosomes 3-
Support Protocol 3: Catalase Assay for 38 (1)
Peroxisomes 3-
3.7 Isolation of Peroxisomes from Tissues and 39 (6)
Cells by Differential and Density Gradient
Centrifugation 3-
Basic Protocol 1: Isolation of a Light 40 (1)
Mitochondrial fraction from Rat Liver 3-
Basic Protocol 2: Isolation of Peroxisomes 41 (1)
from a Rat Liver Light Mitochondrial
Fraction Using a Preformed Continuous
Iodixanol Gradient 3-
Basic Protocol 3: Isolation of Peroxisomes 41 (1)
from a Rat Liver Light Mitochondrial
Fraction Using a Preformed Continuous
Nycodenz Gradient 3-
Basic Protocol 4: Isolation of Peroxisomes 42 (1)
from Yeast Spheroplasts Using a Preformed
Continuous Nycodenz Gradient 3-
Basic Protocol 5: Isolation of Peroxisomes 43 (1)
from Cultured Cells (HepG2) Using a
Preformed Continuous Nycodenz Gradient 3-
Support Protocol: Assay for Endoplasmic 44 (1)
Reticulum Marker Enzyme NADPH-Cytochrome c
Reductase 3-
3.8 Isolation of Nuclei and Nuclear Membranes 45 (7)
from Animal Tissues 3-
Basic Protocol 1: Isolation of Nuclei from 45 (2)
Rat-Liver Homogenate Using a Sucrose
Density Barrier 3-
Alternate Protocol: Isolation of Nuclei 47 (1)
from Animal or Plant (Wheat Germ) Cells
Using an Iodixanol Gradient 3-
Basic Protocol 2: Isolation of Nuclear 47 (1)
Membranes: High-Ionic-Strength Method 3-
Basic Protocol 3: Isolation of Nuclear 48 (1)
Membranes: Low-Ionic-Strength Method 3-
Support Protocol 1: Diphenylamine Assay for 49 (1)
DNA 3-
Support Protocol 2: Orcinol Assay for RNA 3- 50 (1)
Support Protocol 3: Ethidium Bromide Assay 50 (2)
for DNA and RNA 3-
3.9 Isolation of Subcellular Fractions from 52
the Yeast Saccharomyces cerevisiae 3-
Basic Protocol 1: Fractionation of 52 (15)
Spheroplasts by Differential Centrifugation
3-
Support Protocol: Preparation of Yeast 67 (1)
Spheroplasts Using Zymolyase 3-
Basic Protocol 2: Equilibrium Density 68 (2)
Gradient Fractionation Using Nycodenz 3-
Basic Protocol 3: Fractionation of P130 70 (2)
Membranes on Sucrose Step Gradients 3-
Basic Protocol 4: Isolation of Intact 72 (2)
Vacuoles Using Ficoll Step Gradients 3-
Basic Protocol 5: Isolation of Intact 74 (4)
Nuclei with Ficoll Step Gradients 3-
Basic Protocol 6: Isolation of 78 (3)
Lactate-Induced Mitochondria Using Nycodenz
Step Gradients 3-
Basic Protocol 7: Isolation of 81 (3)
Oleate-Induced Peroxisomes Using Sucrose
Step Gradients 3-
Basic Protocol 8: Isolation of Endoplasmic 84 (1)
Reticulum Using Sucrose Step Gradients 3-
Basic Protocol 9: Isolation of Plasma 85 (2)
Membranes from Whole Yeast Cells Using
Sucrose Step Gradients 3-
Basic Protocol 10: Preparation of Cytosol 87
from Whole Yeast Cells 3-
4 Antibodies As Cell Biological Tools
4.1 Production of Monoclonal Antibodies 4- 2 (6)
Basic Protocol 1: Immunization to Produce 2 (1)
Monoclonal Antibodies 4-
Basic Protocol 2: Cell Fusion and Selection 3 (3)
of Hybridomas 4-
Support Protocol 1: Screening Primary 6 (1)
Hybridoma Supernatants 4-
Support Protocol 2: Establishment of 6 (1)
Hybridoma Lines 4-
Support Protocol 3: Cloning by Limiting 7 (1)
Dilution 4-
Support Protocol 4: Preparation of 8 (1)
Cloning/Expansion Medium 4-
4.2 Production of Polyclonal Antisera 4- 8 (4)
Basic Protocol: Immunization to Produce 8 (2)
Polyclonal Antibodies Using Freund's
Adjuvant 4-
Alternate Protocol: Immunization to Produce 10 (1)
Polyclonal Antiserum Using Other Adjuvants
4-
Support Protocol: Preparation of Serum from 11 (1)
Blood 4-
4.3 Purification of Immunoglobulin G 4- 12 (4)
Basic Protocol 1: Ammonium Sulfate 12 (1)
Precipitation and Size-Exclusion
Chromatography 4-
Basic Protocol 2: Affinity Chromatography 13 (1)
Using Protein A-Sepharose 4-
Alternate Protocol 1: Affinity 14 (1)
Chromatography Using Protein G-Sepharose 4-
Alternate Protocol 2: Affinity 15 (1)
Chromatography Using Anti-Rat kappa Chain
Monoclonal Antibody Coupled to Sepharose 4-
Basic Protocol 3: DE52 Ion-Exchange 15 (1)
Chromatography with Tris Cl 4-
4.4 Antibody Conjugates for Cell Biology 4- 16
Basic Protocol: Conjugating Antibodies to 16 (5)
Fluorophores or Biotin 4-
Support Protocol: Methods to Estimate 21
Antibody Concentration 4-
5 Microscopy
5.1 Proper Alignment and Adjustment of the 3 (13)
Light Microscope 5-
Major Components of the Light Microscope 5- 3 (5)
Basic Imaging and Kohler Illumination Light 8 (1)
Paths for Bright-Field and Fluorescence
Microscopy 5-
Basic Protocol 1: Alignment for Kohler 9 (2)
Illumination in Bright-Field, Transmitted
Light Microscopy 5-
Basic Protocol 2: Alignment of the 11 (1)
Eyepieces 5-
Basic Protocol 3: Alignment for Kohler 12 (1)
Illumination in Epifluorescence Microscopy
5-
Basic Protocol 4: Alignment for 13 (2)
Phase-Contrast Microscopy 5-
Support Protocol: Care and Cleaning of 15 (1)
Microscope Optics 5-
5.2 Fluorescence Microscopy 5- 16 (6)
Fluorescence Microscope Optics 5- 16 (1)
Components of the Fluorescence Microscope 5- 17 (4)
The Digital Darkroom 5- 21 (1)
5.3 Immunofluorescence Staining 5- 22 (2)
Basic Protocol: Immunofluorescence Labeling 22 (2)
of Cultured Cells 5-
5.4 Organelle Staining with Fluorescent Dyes 24 (7)
and Fluorescent Lipid Derivatives 5-
Basic Protocol 1: Staining the Endoplasmic 26 (2)
Reticulum in Fixed Cells 5-
Alternate Protocol: Staining the 28 (1)
Endoplasmic Reticulum in Living Cells 5-
Basic Protocol 2: Staining the Golgi 28 (2)
Complex in Living Cells 5-
Basic Protocol 3: Staining Mitochondria 5- 30 (1)
5.5 Basic Confocal Microscopy 5- 31 (7)
Basis of Optical Sectioning 5- 31 (1)
Types of Confocal Microscopes 5- 32 (3)
Practical Guidelines 5- 35 (3)
5.6 Immunoperoxidase Methods for Localization 38 (7)
of Antigens in Cultured Cells and Tissues 5-
Strategic Planning 5- 39 (1)
Basic Protocol 1: Immunoperoxidase Staining 40 (2)
of Cultured Cells 5-
Basic Protocol 2: Immunoperoxidase Staining 42 (3)
of Tissue Sections 5-
5.7 Cryo-Immunogold Electron Microscopy 5- 45 (5)
Basic Protocol: Immunogold Labeling 5- 45 (2)
Support Protocol 1 : Fixation of Cells for 47 (1)
Immunogold Labeling 5-
Support Protocol 2: Fixation of Tissue for 47 (1)
Immunogold Labeling 5-
Support Protocol 3: Cryosectioning for 48 (1)
Immunogold Labeling 5-
Support Protocol 4: Preparation of Carbon- 49 (1)
and Formvar-Coated Copper Grids 5-
5.8 Correlative Video Light/Electron 50 (4)
Microscopy 5-
Basic Protocol: Correlative Video 50 (4)
Light/Electron Microscopy 5-
5.9 Fluorescent Speckle Microscopy (FSM) of 54 (13)
Microtubules and Actin in Living Cells 5-
Strategic Planning 5- 54 (1)
Basic Protocol 1: Designing a Microscope 54 (4)
System for Time-Lapse Digital FSM 5-
Basic Protocol 2: Time-Lapse FSM Imaging of 58 (3)
the Cytoskeleton in Living Cells 5-
Basic Protocol 3: Qualitative and 61 (1)
Quantitative Analysis of Time-Lapse FSM
Image Series 5-
Support Protocol 1: Preparation of 62 (3)
Fluorescently Labeled Tubulin for FSM 5-
Support Protocol 2: Preparation of 65 (2)
Fluorescently Labeled Actin for FSM 5-
5.10 GFP As a Live Cell Imaging Tool 5- 67
Preparing a Fusion Construct 5- 68
6 Characterization of Cellular Proteins
6.1 Analysis of the Association of Proteins 2 (4)
with Membranes 6-
Basic Protocol 1: Alkaline Carbonate 2 (1)
Extraction 6-
Alternate Protocol 1: Urea Extraction 6- 3 (1)
Alternate Protocol 2: High-Salt Extraction 3 (1)
6-
Alternate Protocol 3: Triton X-114 Phase 3 (1)
Separation 6-
Support Protocol 1: Triton X-114 4 (1)
Precondensation 6-
Basic Protocol 2: PI-PLC Cleavage of 5 (1)
GPI-Linked Proteins 6-
Basic Protocol 3: Detergent Solubilization 6 (1)
of Triton X-100 Insoluble Integral Membrane
and GPI-Linked Proteins 6-
6.2 Determination of Molecular Size by Zonal 6
Sedimentation Analysis on Sucrose Density
Gradients 6-
Basic Protocol 1: Zonal Sedimentation Using 7 (2)
Sucrose Gradients Formed by a Gradient
Master 6-
Alternate Protocol 1: Zonal Sedimentation 9 (2)
Using Sucrose Gradients Formed by a
Gradient Maker 6-
Support Protocol 1: Use and Preparation of 11 (1)
Common Molecular Size Markers 6-
Basic Protocol 2: Fractionation by Puncture 12 (1)
and Elution from the Bottom of the Gradient
6-
Alternate Protocol 2: Fractionation by 13 (1)
Peristaltic Elution from the Bottom of the
Gradient 6-
Support Protocol 2: Determination of 14
Sedimentation Coefficients by Extrapolation
from Migration of Standard Proteins 6-
6.3 Determination of Molecular Size by -15 (18)
Size-Exclusion Chromatography (Gel
Filtration) 6-
Strategic Planning 6- 15 (1)
Basic Protocol 1: SE-HPLC 6- 16 (3)
Basic Protocol 2: Conventional SEC 6- 19
7 Electrophoresis and Immunoblotting
7.1 One-Dimensional SDS Gel Electrophoresis 3 (11)
of Proteins 7-
Electricity and Electrophoresis 7- 3 (2)
Basic Protocol 1: Denaturing (SDS) 5 (5)
Discontinuous Gel Electrophoresis: Laemmli
Gel Method 7-
Alternate Protocol 1: Electrophoresis in 10 (1)
Tris-Tricine Buffer Systems 7-
Alternate Protocol 2: Separation of 11 (3)
Proteins on Gradient Gels 7-
7.2 One-Dimensional Electrophoresis Using 14 (5)
Nondenaturing Conditions 7-
Basic Protocol: Continuous Electrophoresis 14 (2)
in Nondenaturing Polyacrylamide Gels 7-
Alternate Protocol: Native Discontinuous 16 (3)
Electrophoresis and Generation of Molecular
Weight Standard Curves (Ferguson Plots) 7-
7.3 Two-Dimensional Gel Electrophoresis 7- 19 (10)
Basic Protocol 1: High-Resolution 20 (3)
Equilibrium Isoelectric Focusing in Tube
Gels 7-
Support Protocol 1: Conducting pH Profile 23 (1)
Measurements 7-
Alternate Protocol 1: Nonequilibrium 23 (1)
Isoelectric Focusing of Very Acidic
Proteins 7-
Alternate Protocol 2: Nonequilibrium 24 (1)
Isoelectric Focusing of Basic Proteins 7-
Support Protocol 2: Preparing Cell Extracts 24 (2)
for Isoelectric Focusing 7-
Basic Protocol 2: Second-Dimension 26 (1)
Electrophoresis of IEF Tube Gels 7-
Support Protocol 3: Preparing Molecular 27 (1)
Weight Standards for Two-Dimensional Gels 7-
Alternate Protocol 3: Diagonal Gel 28 (1)
Electrophoresis (Nonreducing/Reducing Gels)
7-
7.4 One-Dimensional Isoelectric Focusing of 29 (2)
Proteins in Slab Gels 7-
Basic Protocol: Isoelectric Focusing in 29 (1)
Slab Gels Under Denaturing Conditions 7-
Support Protocol: Electroblotting from 30 (1)
Denaturing Isoelectric-Focusing Slab Gels 7-
7.5 Agarose Gel Electrophoresis of Proteins 7- 31 (1)
Basic Protocol: Agarose Gel Electrophoresis 31 (1)
and Blotting with Immunodetection 7-
7.6 Staining Proteins in Gels 7- 32 (6)
Basic Protocol 1: Staining Protein Gels 32 (1)
with Coornassie Blue 7-
Alternate Protocol: Staining Protein Gels 33 (1)
with Coomassic Blue After Isoelectric
Focusing 7-
Basic Protocol 2: Staining Protein Gels 34 (1)
with Silver 7-
Basic Protocol 3: Fluorescence Detection of 35 (2)
Proteins in Gels 7-
Basic Protocol 4: Reversible Protein 37 (1)
Staining with Zinc 7-
7.7 Immunoblotting and Immunodetection 7- 38 (6)
Basic Protocol 1: Protein Blotting with 38 (2)
Tank Transler Systems 7-
Alternate Protocol 1: Protein Blotting with 40 (1)
Semidry Systems 7-
Support Protocol 1: Reversible Staining of 41 (1)
Transferred Proteins 7-
Basic Protocol 2: Immunoprobing with 42 (1)
Directly Conjugated Secondary Antibody 7-
Alternate Protocol 2: Immunoprobing with 42 (1)
Avidin-Biotin Coupling to Secondary
Antibody 7-
Basic Protocol 3: Visualization with 43 (1)
Luminescent Substrates 7-
Support Protocol 2: Stripping and Reusing 44 (1)
Membranes 7-
7.8 Detection and Quantitation of 44
Radiolabeled Proteins in Gels and Blots 7-
Basic Protocol: Autoradiography 7- 44 (2)
Support Protocol 1: Fixing and Drying Gels 46 (1)
for Autoradiography 7-
Support Protocol 2: Use of Intensifying 47 (1)
Screens 7-
Alternate Protocol 1: Fluorography 7- 47 (1)
Support Protocol 3: Densitometry 7- 48 (1)
Alternate Protocol 2: Phosphor Imaging 7- 48
8 Protein Labeling and Immunoprecipitation
8.1 Metabolic Labeling with Radiolabeled 2 (5)
Amino Acids 8-
Safety Precautions for Working with 2 (2)
35S-Labeled Compounds 8-
Basic Protocol: Pulse-Labeling of Cells in 4 (1)
Suspension with [35S]Methionine 8-
Alternate Protocol l: Pulse-Labeling of 4 (1)
Adherent Cells with [35S]Methionine 8-
Alternate Protocol 2: Pulse-Chase Labeling 5 (1)
of Cells with [35S]Methionine 8-
Alternate Protocol 3: Long-Term Labeling of 6 (1)
Cells with [35S]Methionine 8-
Alternate Protocol 4: Metabolic Labeling 6 (1)
with Other Radiolabeled Amino Acids 8-
Support Protocol: TCA Precipitation to 7 (1)
Determine Label Incorporation 8-
8.2 Metabolic Labeling of Glycoproteins with 7 (4)
Radiolabeled Sugars 8-
Basic Protocol: Pulse-Chase Labeling with 8 (1)
Radiolabeled Sugars 8-
Alternate Protocol: Long-Term Labeling with 9 (2)
Radiolabeled Sugars 8-
8.3 Metabolic Labeling with Radiolabeled 11 (2)
Fatty Acids 8-
Basic Protocol: Biosynthetic Labeling with 11 (2)
Fatty Acids 8-
8.4 Radioiodination of Cellular Proteins 8- 13 (4)
Safety Precautions for Working with 13 (1)
125I-Labeled Compounds 8-
Basic Protocol 1: Cell Surface Labeling 13 (1)
with 125I Using Lactoperoxidase 8-
Basic Protocol 2: Radioiodination of 14 (1)
Detergent-Solubilized Membrane Proteins 8-
Support Protocol: Membrane Preparation by 15 (1)
Homogenization 8-
Basic Protocol 3: Lactoperoxidase-Catalyzed 16 (1)
Radioiodination of Soluble Proteins 8-
8.5 Immunoprecipitation 8- 17 (6)
Basic Protocol 1: Immunoprecipitation Using 18 (3)
Cells in Suspension Lysed with a
Nondenaturing Detergent Solution 8-
Alternate Protocol 1: Immunoprecipitation 21 (1)
Using Adherent Cells Lysed with a
Nondenaturing Detergent Solution 8-
Alternate Protocol 2: Immunoprecipitation 22 (1)
Using Cells Lysed with Detergent Under
Denaturing Conditions 8-
Alternate Protocol 3: Immunoprecipitation 22 (1)
Using Cells Lysed Without Detergent 8-
Basic Protocol 2: 23 (1)
Immunoprecipitation-Recapture 8-
8.6 Metabolic Labeling and 23
Immunoprecipitation of Yeast Proteins 8-
Basic Protocol: Labeling and 24 (2)
Immunoprecipitating Yeast Proteins 8-
Alternate Protocol: Making Yeast 26
Spheroplasts 8-
9 Protein Phosphorylation
9.1 Labeling Cultured Cells with 32Pi and 2
Preparing Cell Lysates for
Immunoprecipitation 9-
Basic Protocol: Labeling Cultured Cells 2 (1)
with 32Pi and Lysis Using Mild Detergent 9-
Alternate Protocol: Lysis of Cells by 3
Boiling in SDS 9-
9.2 Immunological Detection of -4 (13)
Phosphorylation 9-
Basic Protocol 1: Immunodetection of 4 (3)
Protein Phosphorylation by Immunoblotting 9-
Basic Protocol 2: Immunodetection of 7 (1)
Protein Phosphorylation by
Immunoprecipitation Followed by
Immunoblotting 9-
Basic Protocol 3: Fluorescent 8 (1)
Immunostaining of Tissue Culture Cells 9-
9.3 Detection of Map Kinase Signaling 9- 9
Basic Protocol l: Determination of Map 10 (2)
Kinase (ERK) Activity by
Immunoprecipitation 9-
Basic Protocol 2: In-Gel Kinase Assay 9- 12 (1)
Basic Protocol 3: JNK Assay 9- 13 (1)
Support Protocol: Preparation of 14
GST-JUN-Glutathione Beads 9-
10 Protein Trafficking
10.1 Use of Glycosidases to Study Protein 2 (4)
Trafficking 10-
Basic Protocol l: Endoglycosidase H 3 (1)
Digestion 10-
Basic Protocol 2: Peptide: N-Glycosidase F 3 (2)
Digestion 10-
Basic Protocol 3: Sialidase (Neuraminidase) 5 (1)
Digestion 10-
Basic Protocol 4: 5 (1)
Endo-alpha-N-Acetylgalactosaminidase
Digestion 10-
10.2 Endocytosis 10- 6 (9)
Basic Protocol 1: Measuring the 7 (1)
Steady-State Surface-to-Internal
Distribution of the Transferrin Receptor 10-
Basic Protocol 2: Measuring the Kinetics of 8 (1)
Transferrin Internalization 10-
Alternate Protocol 1: Measuring the 9 (1)
Kinetics of Membrane Protein
Internalization Using 125I-Labeled
Antibodies 10-
Alternate Protocol 2: Measuring the 10 (1)
Kinetics of Transferrin Internalization in
Cells Grown in Suspension 10-
Basic Protocol 3: Measuring the Kinetics of 11 (1)
Transferrin Receptor Recycling 10-
Support Protocol 1 Iron-Loading Transferrin 12 (1)
10-
Support Protocol 2: Radiolabeling of 12 (1)
Diferric Transferrin 10-
Basic Protocol 4: Measuring Fluid-Phase 13 (1)
Uptake 10-
Support Protocol 3: Inhibition of 14 (1)
Clathrin-Mediated Endocytosis by Potassium
Depletion 10-
Support Protocol 4: Inhibition of 14 (1)
Clathrin-Mediated Endocytosis by Cytosol
Acidification 10-
10.3 Protein Transport to the Plasma Membrane 15
10-
Basic Protocol: Measuring Arrival at the 15 (1)
Cell Surface by Digestion with Sialidase 10-
Alternate Protocol: Measuring Arrival at 16
the Cell Surface by Biotinylation of
Cell-Surface Molecules 10-
10.5 Membrane Traffic in Polarized Epithelial 11
Cells 10-
Basic Protocol 1: Transfection of Polarized 18 (1)
Epithelial Cells in Suspension and
Selection of Resistant Clones 10-
Support Protocol 1: Picking Stably 19 (1)
Transfected Clones 10-
Support Protocol 2: Culture of Epithelial 20 (1)
Cells on Filters 10-
Support Protocol 3: Determining the 21 (1)
Leakiness of a Monolayer of Cells Grown on
a Filter 10-
Basic Protocol t: Pulse-Chase Experiments 22 (1)
in Polarized Epithelial Cells 10-
Basic Protocol 3: Biotinylation of Newly 23 (1)
Synthesized Epithelial Cell Surface
Proteins 10-
Basic Protocol 4: Indirect 24
Immunofluorescence of Proteins in Polarized
Epithelial Cells 10-
11 Cell Proliferation, Cell Aging, and Cellular
Death
11.1 Determining Cell Cycle Stages by Flow 2 (6)
Cytometry 11-
Basic Protocol 1: Cell Cycle Analysis of 2 (2)
Fixed Cells Stained with Propidium Iodide
11-
Alternate Protocol 1: Cell Cycle Analysis 4 (2)
of Fixed Cells Stained with DAPI 11-
Basic Protocol 2: Cell Cycle Analysis of 6 (1)
Unfixed, Detergent-Permeabilized Cells
Stained with PI 11-
Alternate Protocol 2: Cell Cycle Analysis 6 (1)
of Unfixed, Detergent-Permeabilized Cells
Stained with DAPI 11-
Basic Protocol 3: Staining of Live Cells 6 (1)
with Hoechst 33342 11-
Basic Protocol 4: Bivariate Analysis of DNA 7 (1)
Content and Expression of Cyclins D, E, A,
or B1 11-
11.2 Methods for Synchronizing Cells At 8 (8)
Specific Stages of the Cell Cycle 11-
Basic Protocol 1: Enrichment of Mitotic 9 (1)
Cells by Mitotic Shake-Off 11-
Alternate Protocol 1: Pre-Enrichment of 10 (1)
Exponential Cultures for Mitotic Cells 11-
Alternate Protocol 2: Enrichment of Mitotic 10 (1)
Cells by Nocodazole Arrest 11-
Basic Protocol 2: Enrichment of Cells At 10 (1)
G0/G1 by Serum Starvation 11-
Alternate Protocol 3: Enrichment of Cells 11 (1)
At G0/G1 by Amino Acid Starvation 11-
Basic Protocol 3: Enrichment of G1-Phase 11 (1)
Cells Using Lovastatin 11-
Alternate Protocol 4: Enrichment of 12 (1)
G1-Phase Cells by Mimosine Arrest 11-
Basic Protocol 4: Synchronizing Cells At 13 (1)
the Onset of S Phase by Double-Thymidine
Block 11-
Alternate Protocol 5: Performing Sequential 14 (1)
G1/S Blocks 11-
Support Protocol 1: Determining Mitotic 14
Index 11-
Support Protocol 2: Monitoring -15 (31)
[3H]Thymidine Incorporation into DNA by TCA
Precipitation 11-
11.3 Assays for CDK Activity and DNA 16 (5)
Replication in the Cell Cycle 11-
Basic Protocol 1: Measuring CDK Activity 11- 16 (3)
Basic Protocol 2: Measuring DNA Replication 19 (2)
Using Incorporation of BrdU 11-
11.4 Assessment of Apoptosis and Necrosis by 21 (12)
DNA Fragmentation and Morphological Criteria
11-
Morphology Assays 11- 21 (1)
Basic Protocol 1: Measurement of Cell Death 21 (2)
by Trypan Blue Exclusion 11-
Basic Protocol 2: Differential Staining of 23 (1)
Cells 11-
Basic Protocol 3: Hoechst Staining of Cells 24 (1)
11-
Support Protocol: Cytospin Preparation of 24 (1)
Cells for Analysis 11-
Assays for Chromatin Cleavage 11- 25 (1)
Basic Protocol 4: TUNEL Assay for DNA 25 (1)
Fragmentation in Cells 11-
Alternate Protocol 1: TUNEL Assay in 26 (1)
Paraffin-Embedded Sections 11-
Basic Protocol 5: Detection of DNA 27 (1)
Fragmentation in Whole Cells 11-
Alternate Protocol 2: Detection of DNA 28 (1)
Fragmentation in Total Genomic DNA 11-
Alternate Protocol 3: Simple Protocol for 29 (1)
Detection of DNA Fragments 11-
Alternate Protocol 4: Phenol Extraction of 29 (1)
DNA Fragments for Agarose Gel
Electrophoresis 11-
Basic Protocol 6: Quantitative Assay of DNA 30 (1)
Fragmentation 11-
Basic Protocol 7: Detection of 31 (2)
High-Molecular-Weight Chromatin Fragments
by Pulsed-Field Agarose Gel Electrophoresis
11-
11.5 Analysis of Caspase Activation During 33
Apoptosis 11-
Basic Protocol 1: Enzymatic Assays for 33 (2)
Caspase Activity 11-
Basic Protocol 2: Detection of Caspase 35 (1)
Activation by Immunoblotting 11-
Alternate Protocol 1: Cell Lysis with 36 (1)
Guanidine Hydrochloride for Immunoblotting
11-
Support Protocol 1: Removing (Stripping) 37 (1)
Primary and Secondary Antibodies from Blots
11-
Basic Protocol 3: Labeling and Detecting 38 (1)
Active Caspases Using Biotinylated
Substrate Analogs 11-
Alternate Protocol 2: In Vitro Activation 39 (1)
of Caspases in Naive Lysates Followed by
Affinity Labeling 11-
Support Protocol 2: Controls for 40 (1)
Specificity of Affinity-Labeled Active
Caspases 11-
Support Protocol 3: Stripping Membrane in 40
the Presence of d-Biotin for Reprobing with
Antibody 11-
12 In Vitro Reconstitution
12.1 In Vitro Translation 12- 3 (8)
Basic Protocol 1: Production and Use of 3 (2)
mRNA-Dependent Cell-Free Translation System
from Rabbit Reticulocytes 12-
Basic Protocol 2: Production and Use of 5 (2)
mRNA-Dependent Cell-Free Translation System
from Wheat Germ 12-
Basic Protocol 3: In Vitro Protein 7 (1)
Synthesis in Coupled
Transcription/Translation Systems 12-
Support Protocol 1: Production of Uncapped 8 (1)
In Vitro Transcripts 12-
Support Protocol 2: Production of Capped In 9 (1)
Vitro Transcripts 12-
Alternate Protocol: In Vitro Translation 10 (1)
with Biotinylated Amino Acids 12-
Support Protocol 3: Capture of Biotinylated 11 (1)
Proteins with Streptavidin-Agarose 12-
12.2 Cotranslational Translocation of 11 (5)
Proteins into Canine Rough Microsomes 12-
Basic Protocol: Translocation into Canine 12 (1)
Rough Microsomes 12-
Support Protocol l: Preparation of RMs from 13 (2)
Canine Pancreas 12-
Support Protocol 2: Preparation of 15 (1)
EDTA-Stripped Rough Microsomes 12-
Support Protocol 3: Preparation of 15 (1)
Column-Washed Rough Microsomes 12-
12.3 In Vitro Analysis of Endoplasmic 16 (10)
Reticulum-To-Golgi Transport in Mammalian
Cells 12-
Basic Protocol 1: Reconstitution of 16 (2)
ER-to-Golgi Transport in Semi-Intact Cells
12-
Alternate Protocol: Reconstitution of 18 (1)
ER-to-cis-Golgi Transport in Semi-Intact
Cells 12-
Basic Protocol 2: In Vitro Reconstitution 19 (1)
of ER-to-Golgi Transport in Mammalian
Microsomes 12-
Basic Protocol 3: In Vitro Formation and 20 (2)
Isolation of ER-Derived Vesicles 12-
Support Protocol 1: Preparation of 22 (1)
Microsomal Membranes from NRK Cells 12-
Support Protocol 2: Propagation of VSV 23 (1)
ts045 12-
Basic Protocol 4: Fusion of ER-Derived 23 (1)
Vesicles with Golgi Membranes 12-
Support Protocol 3: Preparation of Rat 24 (1)
Liver Cytosol 12-
Support Protocol 4: Preparation of Golgi 25 (1)
Membranes from Rat Liver 12-
12.4 Nuclear Import in 26 (7)
Digitonin-Permeabilized Cells 12-
Basic Protocol: Nuclear Import Assay in 27 (1)
Attached HeLa Cells 12-
Support Protocol 1: Preparation of Xenopus 28 (2)
Ovarian Cytosol 12-
Support Protocol 2: Production of 30 (1)
Fluorescent Import Substrate: TRITC-BSA-NLS
12-
Support Protocol 3: Production of 31 (2)
Fluorescent Recombinant Import Substrate:
GFP-GST-NLS 12-
12.5 Preparation and Use of Interphase 33 (12)
Xenopus Egg Extracts 12-
Basic Protocol 1: Preparation of Interphase 33 (1)
Egg Extracts 12-
Alternate Protocol 1: Preparing 34 (1)
Fractionated Interphase Extracts 12-
Support Protocol 1: Injection of Frogs to 35 (1)
Obtain Eggs 12-
Basic Protocol 2: Nuclear Assembly in the 36 (2)
Interphase Egg Extract 12-
Support Protocol 2: Preparation of 38 (2)
Demembranated Sperm Chromatin to Use as
Templates for Nuclear Assembly 12-
Basic Protocol 3: Nuclear Protein Import In 40 (1)
Vitro 12-
Basic Protocol 4: DNA Replication with 41 (1)
Continuous Labeling 12-
Alternate Protocol 2: Pulse-Labeling DNA to 42 (1)
Assess Replication 12-
Basic Protocol 5: Preparation of Oocyte 43 (1)
Extract 12-
Support Protocol 3: Immunodepletion of 44 (1)
Extracts 12-
Support Protocol 4: Addition of Protein to 45 (1)
Extracts 12-
12.6 Analysis of the Cell Cycle Using Xenopus 45 (8)
Egg Extracts 12-
Basic Protocol 1: Preparation of the 45 (2)
Cycling Extract 12-
Basic Protocol 2: Preparation of 47 (2)
CSF-Arrested Extracts 12-
Basic Protocol 3: Preparing a Mitotic 49 (1)
Extract 12-
Basic Protocol 4: Driving Interphase 49 (1)
Extracts into Mitosis 12-
Alternate Protocol: Generating a 50 (1)
Replication Checkpoint In Vitro 12-
Support Protocol 1: Monitoring the Cell 51 (1)
Cycle State of Extracts 12-
Support Protocol 2: Assaying Histone H1 51 (1)
Kinase Activity 12-
Support Protocol a: Release of CSF-Arrested 52 (1)
Extracts and Their Progression into
Interphase 12-
12.7 Mitotic Spindle Assembly In Vitro 12- 53 (10)
Basic Protocol 1: Analyzing DMSO and 53 (1)
Centrosome Aster Reactions 12-
Basic Protocol 2: Analyzing Sperm DNA 54 (1)
"Half-Spindle" Reactions 12-
Basic Protocol 3: Analyzing Sperm DNA 55 (1)
"Cycling" Reactions 12-
Basic Protocol 4: Analyzing DNA-Bead 56 (1)
Reactions 12-
Support Protocol 1: Preparation of CSF 57 (1)
Extract 12-
Support Protocol 2: Preparation of 58 (1)
Rhodamine-Labeled Tubulin 12-
Support Protocol 3: Motor Disruption 12- 59 (1)
Support Protocol 4: Reaction Spin-Downs 12- 60 (2)
Support Protocol 5: DNA-Coated Beads 12- 62 (1)
12.8 Analysis of Apoptosis Using Xenopus Egg 63 (4)
Extracts 12-
Basic Protocol: Preparation of Apoptotic 63 (1)
Extracts and Assessing Apoptosis 12-
Alternate Protocol: Separating Apoptosis 64 (1)
into Latent and Execution Phases 12-
Support Protocol 1: Measuring Caspase 65 (1)
3-Like Activity 12-
Support Protocol 2: Preparation of 65 (1)
Mitochondria from Xenopus Egg Extracts 12-
Support Protocol 3: Cytochrome c Release 66 (1)
Assays 12-
Support Protocol 4: Cytochrome c Release 66 (1)
Assay Using Purified Mitochondria and
Cytosol 12-
12.9 In Vitro Transcription 12- 67
Basic Protocol: In Vitro Transcription 67 (1)
Reactions with Nuclear Extracts 12-
Support Protocol 1: Preparation of a 68 (2)
Nuclear Extract from HeLa Cells 12-
Support Protocol 2: Preparation of 70 (3)
High-Salt Drosophila Extracts 12-
Support Protocol 3: Preparation of the 73 (1)
Soluble Nuclear Fraction from Isolated
Drosophila Embryo Nuclei 12-
Support Protocol 4: Primer Extension 73
Analyses of In Vitro Transcription Products
12-
13 Cell Adhesion and Extracellular Matrix
13.1 Cell-Substrate Adhesion Assays 13- 3 (4)
Basic Protocol 1: Spreading Assay 13- 4 (1)
Basic Protocol 2: Attachment Assay 13- 5 (1)
Support Protocol: Preparation of 6 (1)
Peptide-Protein Conjugates 13-
13.2 Quantitative Measurement of Cell 7 (2)
Adhesion Using Centrifugal Force 13-
Basic Protocol: Centrifugation Cell 7 (2)
Adhesion Assay 13-
13.3 Cadherin-Dependent Cell-Cell Adhesion 13- 9 (9)
Strategic Planning 13- 10 (1)
Basic Protocol 1: Short-Term Aggregation 10 (2)
Culture 13-
Alternate Protocol: Long-Term Aggregation 12 (1)
Culture 13-
Basic Protocol 2: Mixed-Cell Aggregation 12 (1)
Culture 13-
Support Protocol 1: Dissociation of 13 (1)
Fibroblasts by TC Treatment 13-
Support Protocol 2: Dissociation of 14 (1)
Embryonic Cells by TC Treatment 13-
Support Protocol 3: Dissociation of Cells 15 (1)
by LTE or TE Treatment 13-
Basic Protocol 3: Detection of Cadherins 15 (1)
and Gatenins 13-
Basic Protocol 4: Inhibition of Cadherin 16 (1)
Function 13-
Basic Protocol 5: Restoration of Cadherin 17 (1)
Activity in Cadherin- or Catenin-Deficient
Cell Lines 13-
13.4 Analyzing Integrin-Dependent Adhesion 13- 18 (8)
Basic Protocol 1: Analyzing 18 (2)
Integrin-Dependent Adhesion in Cell-Based
Assays 13-
Basic Protocol 2: Analyzing Integrin-Ligand 20 (1)
Interactions in Solid-Phase Assays 13-
Support Protocol l : Integrin Purification 21 (3)
13-
Support Protocol 2: Coupling of Antibodies 24 (1)
to Sepharose 13-
Support Protocol 3: Biotinylation of 25 (1)
Integrin Ligands 13-
13.5 Analysis of Cell-Cell Contact Mediated 26 (12)
by Ig Superfamily Cell Adhesion Molecules 13-
Basic Protocol 1: Purification of IgSF-CAMS 26 (1)
by Immunoaffinity Chromatography 13-
Support Protocol 1: Preparation of the 27 (1)
Affinity Column 13-
Support Protocol 2: Solubilizing Membrane 27 (2)
Proteins 13-
Basic Protocol 2: Analysis of Protein 29 (1)
Interactions with Fluorescent Microspheres
13-
Support Protocol 3: Coupling Proteins to 30 (1)
Fluorescent Microspheres 13-
Basic Protocol 3: Binding of 30 (1)
Protein-Conjugated Microspheres to Cultured
Cells 13-
Basic Protocol 4: Trans-Interaction Assay 31 (1)
with Myeloma Cells 13-
Support Protocol 4: Stable Transfection of 32 (3)
Myeloma Cells by Protoplast Fusion 13-
Basic Protocol 5: Neurite Outgrowth Assay 35 (1)
13-
Basic Protocol 6: Inhibiting CAM-CAM 36 (1)
Interactions In Vitro 13-
Support Protocol 5: Coating Culture Dishes 37 (1)
with IgSF-CAM 13-
Support Protocol 6: Pre-Coating Glass 37 (1)
Surfaces with Nitrocellulose 13-
13.6 Purification of Fibronectin 13- 38 (4)
Basic Protocol 1: Purification of Plasma 8 (31)
Fibronectin 13-
Basic Protocol 2: Purification of 39 (1)
Fibronectin from Cultured Cells 13-
Alternate Protocol 1: Affinity Purification 40 (1)
of Extracted Cellular Fibronectin 13-
Alternate Protocol 2: Purification of Human 41 (1)
Cellular Fibronectin from Conditioned
Medium 13-
13.7 Purification of Vitronectin 13- 42 (3)
Basic Protocol
13.8 Preparation of Gelled Substrates 13- 45 (3)
Basic Protocol 1: Preparation of Type I 45 (1)
Collagen Substrates 13-
Basic Protocol 2: Preparation of Gelled 46 (1)
Matrigel Substrates 13-
Alternate Protocol 1: Growth of Cells 46 (1)
Inside Matrigel 13-
Alternate Protocol 2: Use of Matrigel In 47 (1)
Vivo for Angiogenic Assays and Tumor Growth
13-
13.9 Preparation of Extracellular Matrices 48 (4)
Produced by Cultured Corneal Endothelial and
PF-HR9 Endodermal Cells 13-
Basic Protocol: Preparation of Bovine 48 (2)
Corneal Endothelial Cell ECM (BCE-ECM) 13-
Alternate Protocol: Preparation of HR9-ECM 50 (1)
13-
Support Protocol 1: Cell Proliferation 51 (1)
Assay 13-
Support Protocol 2: Cell Differentiation 51 (1)
Assay 13-
13.10 Preparation of Extracellular Matrices 52 (6)
Produced by Cultured Fibroblasts 13-
Basic Protocol: Preparation of 52 (2)
Extracellular Matrices Produced by Cultured
Fibroblasts 13-
Support Protocol 1: Cell Attachment Assay 54 (1)
13-
Support Protocol 2: Determination of Cell 55 (1)
Shape 13-
Preparing Two-Dimensional Extracellular 56 (1)
Matrix Controls 13-
Support Protocol 3: Mechanical Compression 56 (2)
of the Fibroblast-Derived Three-Dimensional
Matrix 13-
Support Protocol 4: Solubilization of 58 (1)
Fibroblast-Derived Three-Dimensional Matrix
13-
13.11 Proteoglycan Isolation and Analysis 13- 58 (8)
Basic Protocol 1: Isolation of 59
Proteoglycans from Cultured Cells 13-
Alternate Protocol: Isolation of 9 (51)
Proteoglycan Pools 13-5
Support Protocol: Radiolabeling of 60 (1)
Proteoglycans with 35SO4 or [3H]Glucosamine
13-
Basic Protocol 2: Anion-Exchange 60 (1)
Chromatography Purification of
Proteoglycans with DEAF-Sephacel 13-
Basic Protocol 3: Analysis of Proteoglycans 61 (1)
by Size-Exclusion Chromatography 13-
Basic Protocol 4: Analysis of 62 (1)
Glycosaminoglycan Size Following Alkaline
Elimination 13-
Basic Protocol 5: Analysis of 62 (1)
Glycosaminoglycan Size Following Papain
Digestion 13-
Basic Protocol 6: Analysis of 63 (1)
Glycosaminoglycan Content and Protein Core
by GAG Degradation with Lyases 13-
Basic Protocol 7: Treatment with Nitrous 64 (1)
Acid to Degrade Heparan Sulfate 13-
Basic Protocol 8: Analysis of GAG Type and 64 (1)
Core Protein 13-
Basic Protocol 9: Analysis of GAG Size 13- 65 (1)
Basic Protocol 10: Immunoprecipitation of 65 (1)
Proteoglycans 13-
13.12 Matrix Metalloproteinases 13- 66
Basic Protocol 1: Dissolution and 66 (1)
Degradation of Collagen Fibrils by Live
Cells 13-
Support Protocol: Preparing Rat Tail Tendon 67 (2)
Collagen Type I 13-
Basic Protocol 2: Gelatin/Casein Zymography 69 (2)
13-
Basic Protocol 3: Reverse Zymography 13- 71
14 Cell Motility
14.1 Chemotaxis Assays for Eukaryotic Cells 1 (9)
14-
Strategic Planning 14- 1 (1)
Basic Protocol 1: Filter Assay for 2 (3)
Chemotaxis 14-
Support Protocol 1: Calculating the 5 (1)
Distance Cells Are Expected to Move in
Thick Filters in the Absence of Chemotaxis
14-
Basic Protocol 2: Under-Agarose Chemotaxis 6 (1)
Assay 14-
Basic Protocol 3: Small Population 7 (1)
Chemotaxis Assay 14-
Basic Protocol 4: Bridge Chemotaxis Assay 8 (1)
14-
Basic Protocol 5: Pipet Chemotaxis Assay 14- 9 (1)
14.2 Invasion Assays 14- 10 (2)
Basic Protocol: Measuring Invasion Through 10 (1)
a Matrix 14-
Support Protocol: Preparation of 11 (1)
Matrigel-Coated Filters 14-
14.3 Cell Traction 14- 12 (8)
Basic Protocol 1: Measuring Cell Traction 12 (1)
on Wrinkling Substrates 14-
Support Protocol 1: Calibrating 13 (1)
Microneedles 14-
Alternate Protocol 1: Measuring Cell 14 (1)
Traction on Wrinkling Substrates with an
Alternative Polymer 14-
Alternate Protocol 2: Measuring Cell 14 (1)
Traction on Nonwrinkling Substrates 14-
Support Protocol 2: Preparing a Modified 15 (1)
Airbrush Apparatus 14-
Basic Protocol 2: Measuring Cell Traction 16 (3)
on Micromachined Substrates 14-
Support Protocol 3: Silanizing Coverslips 19 (1)
14-
Support Protocol 4: Preparing a Polarized 19 (1)
Reflection Cube 14-
14.4 Cell Wound Assays 14- 20 (6)
Strategic Planning 14- 20 (1)
Basic Protocol: Wound Detection in Cultured 21 (1)
Monolayers Using Fluorescein Dextran 14-
Alternate Protocol 1: Wound Detection in 22 (2)
Mammalian Tissues Using Fluorescein Dextran
14-
Alternate Protocol 2: Wound Detection Using 24 (2)
Albumin As a Wound Tracer 14-
14.5 Dictyostelium Cell Dynamics 14- 26
Basic Protocol 1: Imaging GFP-Labeled 26 (1)
Proteins in Live Single Cells 14-
Alternate Protocol 1: Imaging GFP-Labeled 27 (1)
Proteins Following a Uniform Increase in
Chemoattractant 14-
Alternate Protocol 2: Imaging GFP-Labeled 28 (1)
Proteins in a Chemoattractant Gradient 14-
Basic Protocol 2: Imaging GFP-Labeled 29 (1)
Proteins in Aggregation Stream and Mound
Preparations 14-
Support Protocol 1: Plasmid Construction 29 (2)
and Transformation 14-
Support Protocol 2: Phenotypic Screening by 31
Development on Non-Nutrient Agar 14-
15 Organelle Motility
15.1 Microtubule/Organelle Motility Assays 15- 1 (11)
Basic Protocol: MT/Organelle Motility 1 (3)
Assays 15-
Support Protocol 1: Preparation of Simple 4 (1)
Perfusion Chambers and Coverslips 15-
Support Protocol 2: Preparation of Sea 5 (2)
Urchin Sperm Axonemes 15-
Support Protocol 3: Preparation of Porcine 7 (3)
Brain Tubulin 15-
Support Protocol 4: Preparation of Rat 10 (1)
Liver Cell Cytosol 15-
Support Protocol 5: Preparation of Rat 11 (1)
Liver Organelle Fractions 15-
15.2 In Vitro Motility Assay to Study 12 (5)
Translocation of Actin by Myosin 15-
Basic Protocol: Analyzing Actin 12 (3)
Translocation by Myosin 15-
Support Protocol 1: Preparation of Flow 15 (1)
Cells 15-
Support Protocol 2: Purification of Actin 15 (1)
15-
Support Protocol 3: Preparation of 16 (1)
Rhodamine Phalloidin-Labeled Actin 15-
15.3 Organelle Motility in Plant Cells: 17 (2)
Imaging Golgi and ER Dynamics with Green
Fluorescent Protein (GFP) 15-
Basic Protocol: Transient Expression for 17 (2)
Visualization of ER and Golgi Probes in
Leaves 15-
15.4 Movement of Nuclei 15- 19
Basic Protocol: Nuclear Motility Assay 15- 20 (2)
Support Protocol 1: Centrosome Isolation 22 (3)
from Lymphocytes 15-
Support Protocol 2: Titration of 25 (1)
Concentrated Centrosomes 15-
Support Protocol 3: Preparing Fractionated 25 (3)
Interphase Extracts for Nuclear Assembly 15-
Support Protocol 4: Preparation of 28 (1)
High-Speed Supernatant (HSS) 15-
Support Protocol 5: Nuclear Assembly Using 28
DNA-Coated Magnetic Beads as Template 15-
Appendices
A1 Reagents and Solutions A1- 1 (1)
A2 Useful Measurements and Data A2- 1 (1)
2A Useful Measurements and Data A2- 1 (2)
2B Compendium of Drugs Commonly Used in Cell 3 (14)
Biology Research A2-
2C Data on Radioisotopes A2- 17 (3)
2D Absorption and Emission Maxima for Common 20 (5)
Fluorophores A2-
2E Centrifuges and Rotors A2- 25
A3 Commonly Used Techniques A3- 1 (1)
3A Spectrophotometric Determination of 1 (4)
Protein Concentration A3-
3B Colorimetric Detection and Quantitation of 5 (14)
Total Protein A3-
3C Dialysis and Concentration of Protein 19 (3)
Solutions A3-
3D Quantification of DNA and RNA with 22 (4)
Absorption and Fluorescence Spectroscopy A3-
3E Enzymatic Amplification of DNA by PCR; 26 (6)
Standard Procedures and Optimization A3-
3F Micro RT-PCR A3- 32
A4 Selected Suppliers of Reagents and Equipment 1
A4-
References
Index
