9780471483397

Preface

xvii

Contributors

xxi

1 Cell Culture

1.1 Basic Techniques for Mammalian Cell Tissue Culture 1-

Basic Protocol: Trypsinizing and Subculturing Cells from a Monolayer 1-

(1)

Alternate Protocol: Passaging Cells in Suspension Culture 1-

(1)

Support Protocol 1: Freezing Human Cells Grown in Monolayer Cultures 1-

(1)

Support Protocol 2: Freezing Cells Grown in Suspension Culture 1-

(1)

Support Protocol 3: Thawing and Recovering Human Cells 1-

(1)

Support Protocol 4: Determining Cell Number and Viability with a Hemacytometer and Trypan Blue Staining 1-

(1)

Support Protocol 5: Preparing Cells for Transport 1-

1.2 Media for Culture of Mammalian Cells 1-

(11)

Basic Protocol 1: Preparation of Serum-Containing Media 1-

(1)

Basic Protocol 2: Preparing Media for Reduced-Serum or Serum-Free Growth 1-

(2)

Basic Protocol 3: Preparation of Selective Media: HAT Medium 1-

(1)

Basic Protocol 4: Growth of Transformed Cells in Soft Agar 1-

(1)

Support Protocol 1: pH Control in Media 1-

(1)

Support Protocol 2: Use of Antibiotics in Media 1-

(1)

1.3 Aseptic Technique for Cell Culture 1.

.12

Basic Protocol 1: Aseptic Technique 1-

(2)

Basic Protocol 2: Use of the Laminar-Flow Hood 1-

(1)

1.4 Sterilization and Filtration 1-

(6)

Basic Protocol 1: Autoclaving Liquids 1-

(2)

Alternate Protocol: Autoclaving Dry Goods 1-

(1)

Basic Protocol 2: Dry-Heat Sterilization and Depyrogenation 1-

(1)

Basic Protocol 3: Use of Disinfectants: 70% Ethanol 1-

(1)

Filter Sterilization of Solutions 1-

(1)

Basic Protocol 4: Vacuum Filtration 1-

(1)

Basic Protocol 5: Small-Volume Positive-Pressure Filtration of Nonaqueous Solutions 1-

(1)

1.5 Assessing and Controlling Microbial Contamination in Cell Cultures 1-

(5)

Basic Protocol 1: Testing for Bacterial and Fungal Contaminants 1-

(2)

Basic Protocol 2: Testing for Mycoplasma Contamination by Direct Culture 1-

(2)

Basic Protocol 3: Use of Antibiotics to Control Microbial Contamination 1-

(1)

1.6 Media and Culture of Yeast 1-

Preparation of Media 1-

(5)

General Considerations for Culturing Yeast 1-

2 Preparation and Isolation of Cells

2.1 Establishment of Fibroblast Cultures 2-

(4)

Basic Protocol

2.2 Preparation and Culture of Human Lymphocytes 2-

(4)

Basic Protocol 1: Preparation of Lymphocytes by Ficoll-Hypaque Gradient Centrifugation 2-

(2)

Basic Protocol 2: Preparation of Monocytes/Macrophages and "Dendritic-Like" Cells from Lymphocyte Populations 2-

(1)

Basic Protocol 3: Positive Selection of T and B Cells by Monoclonal Antibody-Coated Magnetic Beads 2-

(1)

2.3 Preparation of Endothelial Cells from Human Umbilical Vein 2-

(2)

Basic Protocol 2-

(2)

2.4 Generation of Continuously Growing B Cell Lincs by Epstein-Barr Virus Transformation 2-

Basic Protocol 2-

2.5 Laser Capture Microdissection 2-

-13

(15)

Basic Protocol: Isolation of a Pure Cell Population from Tissue Sections 2-

(3)

Support Protocol: Hematoxylin and Eosin Staining of Tissues for LCM 2-

3 Subcellular Fractionation and Isolation of Organelles

3.1 Overview of Cell Fractionation 3-

(8)

Basic Principles of Centrifugation 3-

(1)

Instrumentation 3-

(3)

Fractionation Media 3-

(2)

Evaluation of Fractionation 3-

(1)

Definitive Procedures 3-

(1)

3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains 3-

(7)

Basic Protocol 1: Isolation of Plasma Membrane Sheets 3-

(2)

Support Protocol 1: Assay for Alkaline Phosphodiesterase I Activity 3-

(2)

Basic Protocol 2: Isolation of Plasma Membrane Domains 3-

(1)

Support Protocol 2: Assay for K+-Stimulated p-Nitrophenylphosphatase Activity 3-

(1)

Support Protocol 3: Assay for 5'-Nucleotidase Activity 3-

(1)

Support Protocol 4: Indirect Immunofluorescent Detection of Proteins Associated with Plasma Membrane Sheets 3-

(1)

3.3 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation 3-

(7)

Basic Protocol 1: Rapid Isolation of Golgi Membranes from Rat Liver Using a Sucrose Density Barrier 3-

(1)

Basic Protocol 2: Isolation of Golgi Membranes from a Rat Liver Light Mitochondrial Fraction by Flotation Through a Discontinuous Sucrose Gradient 3-

(2)

Basic Protocol 3: Isolation of Golgi Membranes from Cultured Cells by Flotation Through a Discontinuous Sucrose Gradient 3-

(1)

Basic Protocol 4: Isolation of Golgi Membranes from a Microsomal Fraction of Hepatocytes in a Self-Generated Gradient of Iodixanol 3-

(2)

Support Protocol: Assay for UDP-Galactose Galactosyltransferase 3-

(1)

3.4 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation 3-

(4)

Basic Protocol 1: Isolation of Lysosomes from Rat Liver Using a Self-Generated Percoll Gradient 3-

(2)

Basic Protocol 2: Isolation of Lysosomes from Human HL-60 Cultured Cells Using a Self Generated Percoll Gradient 3-

(1)

Support Protocol 1: Assay for Acid Phosphatase 3-

(1)

Support Protocol 2: Assay for ß-1V Acetylglucosaminidase 3-

(1)

3.5 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation 3-

(5)

Basic Protocol 1: Preparation of the Heavy Mitochondrial Fraction from Rat Liver 3-

(2)

Basic Protocol 2: Large-Scale Preparation of Mitochondria from Bovine Heart 3-

(1)

Basic Protocol 3: Preparation of Mitochondria from Skeletal Muscle 3-

(1)

Basic Protocol 4: Preparation of Mitochondria from Cultured Cells 3-

(1)

Basic Protocol 5: Preparation of Mitochondria from Yeast (Saccharomyces cerevisiae) 3-

(1)

3.6 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation 3-

(6)

Basic Protocol 1: Resolution of a Rat Liver Mitochondrial Fraction in a Continuous Sucrose Gradient 3-

(1)

Basic Protocol 2: Isolation of Mitochondria from Rat Brain Using a Discontinuous Percoll Gradient 3-

(1)

Basic Protocol 3: Resolution of a Mitochondrial Fraction in a Self-Generated Percoll Gradient 3-

(1)

Support Protocol 1: Succinate Dehydrogenase Assay for Mitochondria 3-

(1)

Support Protocol 2: 3-Galactosidase Assay for Lysosomes 3-

(1)

Support Protocol 3: Catalase Assay for Peroxisomes 3-

(1)

3.7 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient Centrifugation 3-

(6)

Basic Protocol 1: Isolation of a Light Mitochondrial fraction from Rat Liver 3-

(1)

Basic Protocol 2: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Preformed Continuous Iodixanol Gradient 3-

(1)

Basic Protocol 3: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Preformed Continuous Nycodenz Gradient 3-

(1)

Basic Protocol 4: Isolation of Peroxisomes from Yeast Spheroplasts Using a Preformed Continuous Nycodenz Gradient 3-

(1)

Basic Protocol 5: Isolation of Peroxisomes from Cultured Cells (HepG2) Using a Preformed Continuous Nycodenz Gradient 3-

(1)

Support Protocol: Assay for Endoplasmic Reticulum Marker Enzyme NADPH-Cytochrome c Reductase 3-

(1)

3.8 Isolation of Nuclei and Nuclear Membranes from Animal Tissues 3-

(7)

Basic Protocol 1: Isolation of Nuclei from Rat-Liver Homogenate Using a Sucrose Density Barrier 3-

(2)

Alternate Protocol: Isolation of Nuclei from Animal or Plant (Wheat Germ) Cells Using an Iodixanol Gradient 3-

(1)

Basic Protocol 2: Isolation of Nuclear Membranes: High-Ionic-Strength Method 3-

(1)

Basic Protocol 3: Isolation of Nuclear Membranes: Low-Ionic-Strength Method 3-

(1)

Support Protocol 1: Diphenylamine Assay for DNA 3-

(1)

Support Protocol 2: Orcinol Assay for RNA 3-

(1)

Support Protocol 3: Ethidium Bromide Assay for DNA and RNA 3-

(2)

3.9 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae 3-

Basic Protocol 1: Fractionation of Spheroplasts by Differential Centrifugation 3-

(15)

Support Protocol: Preparation of Yeast Spheroplasts Using Zymolyase 3-

(1)

Basic Protocol 2: Equilibrium Density Gradient Fractionation Using Nycodenz 3-

(2)

Basic Protocol 3: Fractionation of P130 Membranes on Sucrose Step Gradients 3-

(2)

Basic Protocol 4: Isolation of Intact Vacuoles Using Ficoll Step Gradients 3-

(2)

Basic Protocol 5: Isolation of Intact Nuclei with Ficoll Step Gradients 3-

(4)

Basic Protocol 6: Isolation of Lactate-Induced Mitochondria Using Nycodenz Step Gradients 3-

(3)

Basic Protocol 7: Isolation of Oleate-Induced Peroxisomes Using Sucrose Step Gradients 3-

(3)

Basic Protocol 8: Isolation of Endoplasmic Reticulum Using Sucrose Step Gradients 3-

(1)

Basic Protocol 9: Isolation of Plasma Membranes from Whole Yeast Cells Using Sucrose Step Gradients 3-

(2)

Basic Protocol 10: Preparation of Cytosol from Whole Yeast Cells 3-

4 Antibodies As Cell Biological Tools

4.1 Production of Monoclonal Antibodies 4-

(6)

Basic Protocol 1: Immunization to Produce Monoclonal Antibodies 4-

(1)

Basic Protocol 2: Cell Fusion and Selection of Hybridomas 4-

(3)

Support Protocol 1: Screening Primary Hybridoma Supernatants 4-

(1)

Support Protocol 2: Establishment of Hybridoma Lines 4-

(1)

Support Protocol 3: Cloning by Limiting Dilution 4-

(1)

Support Protocol 4: Preparation of Cloning/Expansion Medium 4-

(1)

4.2 Production of Polyclonal Antisera 4-

(4)

Basic Protocol: Immunization to Produce Polyclonal Antibodies Using Freund's Adjuvant 4-

(2)

Alternate Protocol: Immunization to Produce Polyclonal Antiserum Using Other Adjuvants 4-

(1)

Support Protocol: Preparation of Serum from Blood 4-

(1)

4.3 Purification of Immunoglobulin G 4-

(4)

Basic Protocol 1: Ammonium Sulfate Precipitation and Size-Exclusion Chromatography 4-

(1)

Basic Protocol 2: Affinity Chromatography Using Protein A-Sepharose 4-

(1)

Alternate Protocol 1: Affinity Chromatography Using Protein G-Sepharose 4-

(1)

Alternate Protocol 2: Affinity Chromatography Using Anti-Rat kappa Chain Monoclonal Antibody Coupled to Sepharose 4-

(1)

Basic Protocol 3: DE52 Ion-Exchange Chromatography with Tris Cl 4-

(1)

4.4 Antibody Conjugates for Cell Biology 4-

Basic Protocol: Conjugating Antibodies to Fluorophores or Biotin 4-

(5)

Support Protocol: Methods to Estimate Antibody Concentration 4-

5 Microscopy

5.1 Proper Alignment and Adjustment of the Light Microscope 5-

(13)

Major Components of the Light Microscope 5-

(5)

Basic Imaging and Kohler Illumination Light Paths for Bright-Field and Fluorescence Microscopy 5-

(1)

Basic Protocol 1: Alignment for Kohler Illumination in Bright-Field, Transmitted Light Microscopy 5-

(2)

Basic Protocol 2: Alignment of the Eyepieces 5-

(1)

Basic Protocol 3: Alignment for Kohler Illumination in Epifluorescence Microscopy 5-

(1)

Basic Protocol 4: Alignment for Phase-Contrast Microscopy 5-

(2)

Support Protocol: Care and Cleaning of Microscope Optics 5-

(1)

5.2 Fluorescence Microscopy 5-

(6)

Fluorescence Microscope Optics 5-

(1)

Components of the Fluorescence Microscope 5-

(4)

The Digital Darkroom 5-

(1)

5.3 Immunofluorescence Staining 5-

(2)

Basic Protocol: Immunofluorescence Labeling of Cultured Cells 5-

(2)

5.4 Organelle Staining with Fluorescent Dyes and Fluorescent Lipid Derivatives 5-

(7)

Basic Protocol 1: Staining the Endoplasmic Reticulum in Fixed Cells 5-

(2)

Alternate Protocol: Staining the Endoplasmic Reticulum in Living Cells 5-

(1)

Basic Protocol 2: Staining the Golgi Complex in Living Cells 5-

(2)

Basic Protocol 3: Staining Mitochondria 5-

(1)

5.5 Basic Confocal Microscopy 5-

(7)

Basis of Optical Sectioning 5-

(1)

Types of Confocal Microscopes 5-

(3)

Practical Guidelines 5-

(3)

5.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues 5-

(7)

Strategic Planning 5-

(1)

Basic Protocol 1: Immunoperoxidase Staining of Cultured Cells 5-

(2)

Basic Protocol 2: Immunoperoxidase Staining of Tissue Sections 5-

(3)

5.7 Cryo-Immunogold Electron Microscopy 5-

(5)

Basic Protocol: Immunogold Labeling 5-

(2)

Support Protocol 1 : Fixation of Cells for Immunogold Labeling 5-

(1)

Support Protocol 2: Fixation of Tissue for Immunogold Labeling 5-

(1)

Support Protocol 3: Cryosectioning for Immunogold Labeling 5-

(1)

Support Protocol 4: Preparation of Carbon- and Formvar-Coated Copper Grids 5-

(1)

5.8 Correlative Video Light/Electron Microscopy 5-

(4)

Basic Protocol: Correlative Video Light/Electron Microscopy 5-

(4)

5.9 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells 5-

(13)

Strategic Planning 5-

(1)

Basic Protocol 1: Designing a Microscope System for Time-Lapse Digital FSM 5-

(4)

Basic Protocol 2: Time-Lapse FSM Imaging of the Cytoskeleton in Living Cells 5-

(3)

Basic Protocol 3: Qualitative and Quantitative Analysis of Time-Lapse FSM Image Series 5-

(1)

Support Protocol 1: Preparation of Fluorescently Labeled Tubulin for FSM 5-

(3)

Support Protocol 2: Preparation of Fluorescently Labeled Actin for FSM 5-

(2)

5.10 GFP As a Live Cell Imaging Tool 5-

Preparing a Fusion Construct 5-

6 Characterization of Cellular Proteins

6.1 Analysis of the Association of Proteins with Membranes 6-

(4)

Basic Protocol 1: Alkaline Carbonate Extraction 6-

(1)

Alternate Protocol 1: Urea Extraction 6-

(1)

Alternate Protocol 2: High-Salt Extraction 6-

(1)

Alternate Protocol 3: Triton X-114 Phase Separation 6-

(1)

Support Protocol 1: Triton X-114 Precondensation 6-

(1)

Basic Protocol 2: PI-PLC Cleavage of GPI-Linked Proteins 6-

(1)

Basic Protocol 3: Detergent Solubilization of Triton X-100 Insoluble Integral Membrane and GPI-Linked Proteins 6-

(1)

6.2 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients 6-

Basic Protocol 1: Zonal Sedimentation Using Sucrose Gradients Formed by a Gradient Master 6-

(2)

Alternate Protocol 1: Zonal Sedimentation Using Sucrose Gradients Formed by a Gradient Maker 6-

(2)

Support Protocol 1: Use and Preparation of Common Molecular Size Markers 6-

(1)

Basic Protocol 2: Fractionation by Puncture and Elution from the Bottom of the Gradient 6-

(1)

Alternate Protocol 2: Fractionation by Peristaltic Elution from the Bottom of the Gradient 6-

(1)

Support Protocol 2: Determination of Sedimentation Coefficients by Extrapolation from Migration of Standard Proteins 6-

6.3 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration) 6-

-15

(18)

Strategic Planning 6-

(1)

Basic Protocol 1: SE-HPLC 6-

(3)

Basic Protocol 2: Conventional SEC 6-

7 Electrophoresis and Immunoblotting

7.1 One-Dimensional SDS Gel Electrophoresis of Proteins 7-

(11)

Electricity and Electrophoresis 7-

(2)

Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method 7-

(5)

Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems 7-

(1)

Alternate Protocol 2: Separation of Proteins on Gradient Gels 7-

(3)

7.2 One-Dimensional Electrophoresis Using Nondenaturing Conditions 7-

(5)

Basic Protocol: Continuous Electrophoresis in Nondenaturing Polyacrylamide Gels 7-

(2)

Alternate Protocol: Native Discontinuous Electrophoresis and Generation of Molecular Weight Standard Curves (Ferguson Plots) 7-

(3)

7.3 Two-Dimensional Gel Electrophoresis 7-

(10)

Basic Protocol 1: High-Resolution Equilibrium Isoelectric Focusing in Tube Gels 7-

(3)

Support Protocol 1: Conducting pH Profile Measurements 7-

(1)

Alternate Protocol 1: Nonequilibrium Isoelectric Focusing of Very Acidic Proteins 7-

(1)

Alternate Protocol 2: Nonequilibrium Isoelectric Focusing of Basic Proteins 7-

(1)

Support Protocol 2: Preparing Cell Extracts for Isoelectric Focusing 7-

(2)

Basic Protocol 2: Second-Dimension Electrophoresis of IEF Tube Gels 7-

(1)

Support Protocol 3: Preparing Molecular Weight Standards for Two-Dimensional Gels 7-

(1)

Alternate Protocol 3: Diagonal Gel Electrophoresis (Nonreducing/Reducing Gels) 7-

(1)

7.4 One-Dimensional Isoelectric Focusing of Proteins in Slab Gels 7-

(2)

Basic Protocol: Isoelectric Focusing in Slab Gels Under Denaturing Conditions 7-

(1)

Support Protocol: Electroblotting from Denaturing Isoelectric-Focusing Slab Gels 7-

(1)

7.5 Agarose Gel Electrophoresis of Proteins 7-

(1)

Basic Protocol: Agarose Gel Electrophoresis and Blotting with Immunodetection 7-

(1)

7.6 Staining Proteins in Gels 7-

(6)

Basic Protocol 1: Staining Protein Gels with Coornassie Blue 7-

(1)

Alternate Protocol: Staining Protein Gels with Coomassic Blue After Isoelectric Focusing 7-

(1)

Basic Protocol 2: Staining Protein Gels with Silver 7-

(1)

Basic Protocol 3: Fluorescence Detection of Proteins in Gels 7-

(2)

Basic Protocol 4: Reversible Protein Staining with Zinc 7-

(1)

7.7 Immunoblotting and Immunodetection 7-

(6)

Basic Protocol 1: Protein Blotting with Tank Transler Systems 7-

(2)

Alternate Protocol 1: Protein Blotting with Semidry Systems 7-

(1)

Support Protocol 1: Reversible Staining of Transferred Proteins 7-

(1)

Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody 7-

(1)

Alternate Protocol 2: Immunoprobing with Avidin-Biotin Coupling to Secondary Antibody 7-

(1)

Basic Protocol 3: Visualization with Luminescent Substrates 7-

(1)

Support Protocol 2: Stripping and Reusing Membranes 7-

(1)

7.8 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 7-

Basic Protocol: Autoradiography 7-

(2)

Support Protocol 1: Fixing and Drying Gels for Autoradiography 7-

(1)

Support Protocol 2: Use of Intensifying Screens 7-

(1)

Alternate Protocol 1: Fluorography 7-

(1)

Support Protocol 3: Densitometry 7-

(1)

Alternate Protocol 2: Phosphor Imaging 7-

8 Protein Labeling and Immunoprecipitation

8.1 Metabolic Labeling with Radiolabeled Amino Acids 8-

(5)

Safety Precautions for Working with 35S-Labeled Compounds 8-

(2)

Basic Protocol: Pulse-Labeling of Cells in Suspension with [35S]Methionine 8-

(1)

Alternate Protocol l: Pulse-Labeling of Adherent Cells with [35S]Methionine 8-

(1)

Alternate Protocol 2: Pulse-Chase Labeling of Cells with [35S]Methionine 8-

(1)

Alternate Protocol 3: Long-Term Labeling of Cells with [35S]Methionine 8-

(1)

Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids 8-

(1)

Support Protocol: TCA Precipitation to Determine Label Incorporation 8-

(1)

8.2 Metabolic Labeling of Glycoproteins with Radiolabeled Sugars 8-

(4)

Basic Protocol: Pulse-Chase Labeling with Radiolabeled Sugars 8-

(1)

Alternate Protocol: Long-Term Labeling with Radiolabeled Sugars 8-

(2)

8.3 Metabolic Labeling with Radiolabeled Fatty Acids 8-

(2)

Basic Protocol: Biosynthetic Labeling with Fatty Acids 8-

(2)

8.4 Radioiodination of Cellular Proteins 8-

(4)

Safety Precautions for Working with 125I-Labeled Compounds 8-

(1)

Basic Protocol 1: Cell Surface Labeling with 125I Using Lactoperoxidase 8-

(1)

Basic Protocol 2: Radioiodination of Detergent-Solubilized Membrane Proteins 8-

(1)

Support Protocol: Membrane Preparation by Homogenization 8-

(1)

Basic Protocol 3: Lactoperoxidase-Catalyzed Radioiodination of Soluble Proteins 8-

(1)

8.5 Immunoprecipitation 8-

(6)

Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution 8-

(3)

Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution 8-

(1)

Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions 8-

(1)

Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent 8-

(1)

Basic Protocol 2: Immunoprecipitation-Recapture 8-

(1)

8.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins 8-

Basic Protocol: Labeling and Immunoprecipitating Yeast Proteins 8-

(2)

Alternate Protocol: Making Yeast Spheroplasts 8-

9 Protein Phosphorylation

9.1 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation 9-

Basic Protocol: Labeling Cultured Cells with 32Pi and Lysis Using Mild Detergent 9-

(1)

Alternate Protocol: Lysis of Cells by Boiling in SDS 9-

9.2 Immunological Detection of Phosphorylation 9-

-4

(13)

Basic Protocol 1: Immunodetection of Protein Phosphorylation by Immunoblotting 9-

(3)

Basic Protocol 2: Immunodetection of Protein Phosphorylation by Immunoprecipitation Followed by Immunoblotting 9-

(1)

Basic Protocol 3: Fluorescent Immunostaining of Tissue Culture Cells 9-

(1)

9.3 Detection of Map Kinase Signaling 9-

Basic Protocol l: Determination of Map Kinase (ERK) Activity by Immunoprecipitation 9-

(2)

Basic Protocol 2: In-Gel Kinase Assay 9-

(1)

Basic Protocol 3: JNK Assay 9-

(1)

Support Protocol: Preparation of GST-JUN-Glutathione Beads 9-

10 Protein Trafficking

10.1 Use of Glycosidases to Study Protein Trafficking 10-

(4)

Basic Protocol l: Endoglycosidase H Digestion 10-

(1)

Basic Protocol 2: Peptide: N-Glycosidase F Digestion 10-

(2)

Basic Protocol 3: Sialidase (Neuraminidase) Digestion 10-

(1)

Basic Protocol 4: Endo-alpha-N-Acetylgalactosaminidase Digestion 10-

(1)

10.2 Endocytosis 10-

(9)

Basic Protocol 1: Measuring the Steady-State Surface-to-Internal Distribution of the Transferrin Receptor 10-

(1)

Basic Protocol 2: Measuring the Kinetics of Transferrin Internalization 10-

(1)

Alternate Protocol 1: Measuring the Kinetics of Membrane Protein Internalization Using 125I-Labeled Antibodies 10-

(1)

Alternate Protocol 2: Measuring the Kinetics of Transferrin Internalization in Cells Grown in Suspension 10-

(1)

Basic Protocol 3: Measuring the Kinetics of Transferrin Receptor Recycling 10-

(1)

Support Protocol 1 Iron-Loading Transferrin 10-

(1)

Support Protocol 2: Radiolabeling of Diferric Transferrin 10-

(1)

Basic Protocol 4: Measuring Fluid-Phase Uptake 10-

(1)

Support Protocol 3: Inhibition of Clathrin-Mediated Endocytosis by Potassium Depletion 10-

(1)

Support Protocol 4: Inhibition of Clathrin-Mediated Endocytosis by Cytosol Acidification 10-

(1)

10.3 Protein Transport to the Plasma Membrane 10-

Basic Protocol: Measuring Arrival at the Cell Surface by Digestion with Sialidase 10-

(1)

Alternate Protocol: Measuring Arrival at the Cell Surface by Biotinylation of Cell-Surface Molecules 10-

10.5 Membrane Traffic in Polarized Epithelial Cells 10-

Basic Protocol 1: Transfection of Polarized Epithelial Cells in Suspension and Selection of Resistant Clones 10-

(1)

Support Protocol 1: Picking Stably Transfected Clones 10-

(1)

Support Protocol 2: Culture of Epithelial Cells on Filters 10-

(1)

Support Protocol 3: Determining the Leakiness of a Monolayer of Cells Grown on a Filter 10-

(1)

Basic Protocol t: Pulse-Chase Experiments in Polarized Epithelial Cells 10-

(1)

Basic Protocol 3: Biotinylation of Newly Synthesized Epithelial Cell Surface Proteins 10-

(1)

Basic Protocol 4: Indirect Immunofluorescence of Proteins in Polarized Epithelial Cells 10-

11 Cell Proliferation, Cell Aging, and Cellular Death

11.1 Determining Cell Cycle Stages by Flow Cytometry 11-

(6)

Basic Protocol 1: Cell Cycle Analysis of Fixed Cells Stained with Propidium Iodide 11-

(2)

Alternate Protocol 1: Cell Cycle Analysis of Fixed Cells Stained with DAPI 11-

(2)

Basic Protocol 2: Cell Cycle Analysis of Unfixed, Detergent-Permeabilized Cells Stained with PI 11-

(1)

Alternate Protocol 2: Cell Cycle Analysis of Unfixed, Detergent-Permeabilized Cells Stained with DAPI 11-

(1)

Basic Protocol 3: Staining of Live Cells with Hoechst 33342 11-

(1)

Basic Protocol 4: Bivariate Analysis of DNA Content and Expression of Cyclins D, E, A, or B1 11-

(1)

11.2 Methods for Synchronizing Cells At Specific Stages of the Cell Cycle 11-

(8)

Basic Protocol 1: Enrichment of Mitotic Cells by Mitotic Shake-Off 11-

(1)

Alternate Protocol 1: Pre-Enrichment of Exponential Cultures for Mitotic Cells 11-

(1)

Alternate Protocol 2: Enrichment of Mitotic Cells by Nocodazole Arrest 11-

(1)

Basic Protocol 2: Enrichment of Cells At G0/G1 by Serum Starvation 11-

(1)

Alternate Protocol 3: Enrichment of Cells At G0/G1 by Amino Acid Starvation 11-

(1)

Basic Protocol 3: Enrichment of G1-Phase Cells Using Lovastatin 11-

(1)

Alternate Protocol 4: Enrichment of G1-Phase Cells by Mimosine Arrest 11-

(1)

Basic Protocol 4: Synchronizing Cells At the Onset of S Phase by Double-Thymidine Block 11-

(1)

Alternate Protocol 5: Performing Sequential G1/S Blocks 11-

(1)

Support Protocol 1: Determining Mitotic Index 11-

Support Protocol 2: Monitoring [3H]Thymidine Incorporation into DNA by TCA Precipitation 11-

-15

(31)

11.3 Assays for CDK Activity and DNA Replication in the Cell Cycle 11-

(5)

Basic Protocol 1: Measuring CDK Activity 11-

(3)

Basic Protocol 2: Measuring DNA Replication Using Incorporation of BrdU 11-

(2)

11.4 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria 11-

(12)

Morphology Assays 11-

(1)

Basic Protocol 1: Measurement of Cell Death by Trypan Blue Exclusion 11-

(2)

Basic Protocol 2: Differential Staining of Cells 11-

(1)

Basic Protocol 3: Hoechst Staining of Cells 11-

(1)

Support Protocol: Cytospin Preparation of Cells for Analysis 11-

(1)

Assays for Chromatin Cleavage 11-

(1)

Basic Protocol 4: TUNEL Assay for DNA Fragmentation in Cells 11-

(1)

Alternate Protocol 1: TUNEL Assay in Paraffin-Embedded Sections 11-

(1)

Basic Protocol 5: Detection of DNA Fragmentation in Whole Cells 11-

(1)

Alternate Protocol 2: Detection of DNA Fragmentation in Total Genomic DNA 11-

(1)

Alternate Protocol 3: Simple Protocol for Detection of DNA Fragments 11-

(1)

Alternate Protocol 4: Phenol Extraction of DNA Fragments for Agarose Gel Electrophoresis 11-

(1)

Basic Protocol 6: Quantitative Assay of DNA Fragmentation 11-

(1)

Basic Protocol 7: Detection of High-Molecular-Weight Chromatin Fragments by Pulsed-Field Agarose Gel Electrophoresis 11-

(2)

11.5 Analysis of Caspase Activation During Apoptosis 11-

Basic Protocol 1: Enzymatic Assays for Caspase Activity 11-

(2)

Basic Protocol 2: Detection of Caspase Activation by Immunoblotting 11-

(1)

Alternate Protocol 1: Cell Lysis with Guanidine Hydrochloride for Immunoblotting 11-

(1)

Support Protocol 1: Removing (Stripping) Primary and Secondary Antibodies from Blots 11-

(1)

Basic Protocol 3: Labeling and Detecting Active Caspases Using Biotinylated Substrate Analogs 11-

(1)

Alternate Protocol 2: In Vitro Activation of Caspases in Naive Lysates Followed by Affinity Labeling 11-

(1)

Support Protocol 2: Controls for Specificity of Affinity-Labeled Active Caspases 11-

(1)

Support Protocol 3: Stripping Membrane in the Presence of d-Biotin for Reprobing with Antibody 11-

12 In Vitro Reconstitution

12.1 In Vitro Translation 12-

(8)

Basic Protocol 1: Production and Use of mRNA-Dependent Cell-Free Translation System from Rabbit Reticulocytes 12-

(2)

Basic Protocol 2: Production and Use of mRNA-Dependent Cell-Free Translation System from Wheat Germ 12-

(2)

Basic Protocol 3: In Vitro Protein Synthesis in Coupled Transcription/Translation Systems 12-

(1)

Support Protocol 1: Production of Uncapped In Vitro Transcripts 12-

(1)

Support Protocol 2: Production of Capped In Vitro Transcripts 12-

(1)

Alternate Protocol: In Vitro Translation with Biotinylated Amino Acids 12-

(1)

Support Protocol 3: Capture of Biotinylated Proteins with Streptavidin-Agarose 12-

(1)

12.2 Cotranslational Translocation of Proteins into Canine Rough Microsomes 12-

(5)

Basic Protocol: Translocation into Canine Rough Microsomes 12-

(1)

Support Protocol l: Preparation of RMs from Canine Pancreas 12-

(2)

Support Protocol 2: Preparation of EDTA-Stripped Rough Microsomes 12-

(1)

Support Protocol 3: Preparation of Column-Washed Rough Microsomes 12-

(1)

12.3 In Vitro Analysis of Endoplasmic Reticulum-To-Golgi Transport in Mammalian Cells 12-

(10)

Basic Protocol 1: Reconstitution of ER-to-Golgi Transport in Semi-Intact Cells 12-

(2)

Alternate Protocol: Reconstitution of ER-to-cis-Golgi Transport in Semi-Intact Cells 12-

(1)

Basic Protocol 2: In Vitro Reconstitution of ER-to-Golgi Transport in Mammalian Microsomes 12-

(1)

Basic Protocol 3: In Vitro Formation and Isolation of ER-Derived Vesicles 12-

(2)

Support Protocol 1: Preparation of Microsomal Membranes from NRK Cells 12-

(1)

Support Protocol 2: Propagation of VSV ts045 12-

(1)

Basic Protocol 4: Fusion of ER-Derived Vesicles with Golgi Membranes 12-

(1)

Support Protocol 3: Preparation of Rat Liver Cytosol 12-

(1)

Support Protocol 4: Preparation of Golgi Membranes from Rat Liver 12-

(1)

12.4 Nuclear Import in Digitonin-Permeabilized Cells 12-

(7)

Basic Protocol: Nuclear Import Assay in Attached HeLa Cells 12-

(1)

Support Protocol 1: Preparation of Xenopus Ovarian Cytosol 12-

(2)

Support Protocol 2: Production of Fluorescent Import Substrate: TRITC-BSA-NLS 12-

(1)

Support Protocol 3: Production of Fluorescent Recombinant Import Substrate: GFP-GST-NLS 12-

(2)

12.5 Preparation and Use of Interphase Xenopus Egg Extracts 12-

(12)

Basic Protocol 1: Preparation of Interphase Egg Extracts 12-

(1)

Alternate Protocol 1: Preparing Fractionated Interphase Extracts 12-

(1)

Support Protocol 1: Injection of Frogs to Obtain Eggs 12-

(1)

Basic Protocol 2: Nuclear Assembly in the Interphase Egg Extract 12-

(2)

Support Protocol 2: Preparation of Demembranated Sperm Chromatin to Use as Templates for Nuclear Assembly 12-

(2)

Basic Protocol 3: Nuclear Protein Import In Vitro 12-

(1)

Basic Protocol 4: DNA Replication with Continuous Labeling 12-

(1)

Alternate Protocol 2: Pulse-Labeling DNA to Assess Replication 12-

(1)

Basic Protocol 5: Preparation of Oocyte Extract 12-

(1)

Support Protocol 3: Immunodepletion of Extracts 12-

(1)

Support Protocol 4: Addition of Protein to Extracts 12-

(1)

12.6 Analysis of the Cell Cycle Using Xenopus Egg Extracts 12-

(8)

Basic Protocol 1: Preparation of the Cycling Extract 12-

(2)

Basic Protocol 2: Preparation of CSF-Arrested Extracts 12-

(2)

Basic Protocol 3: Preparing a Mitotic Extract 12-

(1)

Basic Protocol 4: Driving Interphase Extracts into Mitosis 12-

(1)

Alternate Protocol: Generating a Replication Checkpoint In Vitro 12-

(1)

Support Protocol 1: Monitoring the Cell Cycle State of Extracts 12-

(1)

Support Protocol 2: Assaying Histone H1 Kinase Activity 12-

(1)

Support Protocol a: Release of CSF-Arrested Extracts and Their Progression into Interphase 12-

(1)

12.7 Mitotic Spindle Assembly In Vitro 12-

(10)

Basic Protocol 1: Analyzing DMSO and Centrosome Aster Reactions 12-

(1)

Basic Protocol 2: Analyzing Sperm DNA "Half-Spindle" Reactions 12-

(1)

Basic Protocol 3: Analyzing Sperm DNA "Cycling" Reactions 12-

(1)

Basic Protocol 4: Analyzing DNA-Bead Reactions 12-

(1)

Support Protocol 1: Preparation of CSF Extract 12-

(1)

Support Protocol 2: Preparation of Rhodamine-Labeled Tubulin 12-

(1)

Support Protocol 3: Motor Disruption 12-

(1)

Support Protocol 4: Reaction Spin-Downs 12-

(2)

Support Protocol 5: DNA-Coated Beads 12-

(1)

12.8 Analysis of Apoptosis Using Xenopus Egg Extracts 12-

(4)

Basic Protocol: Preparation of Apoptotic Extracts and Assessing Apoptosis 12-

(1)

Alternate Protocol: Separating Apoptosis into Latent and Execution Phases 12-

(1)

Support Protocol 1: Measuring Caspase 3-Like Activity 12-

(1)

Support Protocol 2: Preparation of Mitochondria from Xenopus Egg Extracts 12-

(1)

Support Protocol 3: Cytochrome c Release Assays 12-

(1)

Support Protocol 4: Cytochrome c Release Assay Using Purified Mitochondria and Cytosol 12-

(1)

12.9 In Vitro Transcription 12-

Basic Protocol: In Vitro Transcription Reactions with Nuclear Extracts 12-

(1)

Support Protocol 1: Preparation of a Nuclear Extract from HeLa Cells 12-

(2)

Support Protocol 2: Preparation of High-Salt Drosophila Extracts 12-

(3)

Support Protocol 3: Preparation of the Soluble Nuclear Fraction from Isolated Drosophila Embryo Nuclei 12-

(1)

Support Protocol 4: Primer Extension Analyses of In Vitro Transcription Products 12-

13 Cell Adhesion and Extracellular Matrix

13.1 Cell-Substrate Adhesion Assays 13-

(4)

Basic Protocol 1: Spreading Assay 13-

(1)

Basic Protocol 2: Attachment Assay 13-

(1)

Support Protocol: Preparation of Peptide-Protein Conjugates 13-

(1)

13.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force 13-

(2)

Basic Protocol: Centrifugation Cell Adhesion Assay 13-

(2)

13.3 Cadherin-Dependent Cell-Cell Adhesion 13-

(9)

Strategic Planning 13-

(1)

Basic Protocol 1: Short-Term Aggregation Culture 13-

(2)

Alternate Protocol: Long-Term Aggregation Culture 13-

(1)

Basic Protocol 2: Mixed-Cell Aggregation Culture 13-

(1)

Support Protocol 1: Dissociation of Fibroblasts by TC Treatment 13-

(1)

Support Protocol 2: Dissociation of Embryonic Cells by TC Treatment 13-

(1)

Support Protocol 3: Dissociation of Cells by LTE or TE Treatment 13-

(1)

Basic Protocol 3: Detection of Cadherins and Gatenins 13-

(1)

Basic Protocol 4: Inhibition of Cadherin Function 13-

(1)

Basic Protocol 5: Restoration of Cadherin Activity in Cadherin- or Catenin-Deficient Cell Lines 13-

(1)

13.4 Analyzing Integrin-Dependent Adhesion 13-

(8)

Basic Protocol 1: Analyzing Integrin-Dependent Adhesion in Cell-Based Assays 13-

(2)

Basic Protocol 2: Analyzing Integrin-Ligand Interactions in Solid-Phase Assays 13-

(1)

Support Protocol l : Integrin Purification 13-

(3)

Support Protocol 2: Coupling of Antibodies to Sepharose 13-

(1)

Support Protocol 3: Biotinylation of Integrin Ligands 13-

(1)

13.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules 13-

(12)

Basic Protocol 1: Purification of IgSF-CAMS by Immunoaffinity Chromatography 13-

(1)

Support Protocol 1: Preparation of the Affinity Column 13-

(1)

Support Protocol 2: Solubilizing Membrane Proteins 13-

(2)

Basic Protocol 2: Analysis of Protein Interactions with Fluorescent Microspheres 13-

(1)

Support Protocol 3: Coupling Proteins to Fluorescent Microspheres 13-

(1)

Basic Protocol 3: Binding of Protein-Conjugated Microspheres to Cultured Cells 13-

(1)

Basic Protocol 4: Trans-Interaction Assay with Myeloma Cells 13-

(1)

Support Protocol 4: Stable Transfection of Myeloma Cells by Protoplast Fusion 13-

(3)

Basic Protocol 5: Neurite Outgrowth Assay 13-

(1)

Basic Protocol 6: Inhibiting CAM-CAM Interactions In Vitro 13-

(1)

Support Protocol 5: Coating Culture Dishes with IgSF-CAM 13-

(1)

Support Protocol 6: Pre-Coating Glass Surfaces with Nitrocellulose 13-

(1)

13.6 Purification of Fibronectin 13-

(4)

Basic Protocol 1: Purification of Plasma Fibronectin 13-

(31)

Basic Protocol 2: Purification of Fibronectin from Cultured Cells 13-

(1)

Alternate Protocol 1: Affinity Purification of Extracted Cellular Fibronectin 13-

(1)

Alternate Protocol 2: Purification of Human Cellular Fibronectin from Conditioned Medium 13-

(1)

13.7 Purification of Vitronectin 13-

(3)

Basic Protocol

13.8 Preparation of Gelled Substrates 13-

(3)

Basic Protocol 1: Preparation of Type I Collagen Substrates 13-

(1)

Basic Protocol 2: Preparation of Gelled Matrigel Substrates 13-

(1)

Alternate Protocol 1: Growth of Cells Inside Matrigel 13-

(1)

Alternate Protocol 2: Use of Matrigel In Vivo for Angiogenic Assays and Tumor Growth 13-

(1)

13.9 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9 Endodermal Cells 13-

(4)

Basic Protocol: Preparation of Bovine Corneal Endothelial Cell ECM (BCE-ECM) 13-

(2)

Alternate Protocol: Preparation of HR9-ECM 13-

(1)

Support Protocol 1: Cell Proliferation Assay 13-

(1)

Support Protocol 2: Cell Differentiation Assay 13-

(1)

13.10 Preparation of Extracellular Matrices Produced by Cultured Fibroblasts 13-

(6)

Basic Protocol: Preparation of Extracellular Matrices Produced by Cultured Fibroblasts 13-

(2)

Support Protocol 1: Cell Attachment Assay 13-

(1)

Support Protocol 2: Determination of Cell Shape 13-

(1)

Preparing Two-Dimensional Extracellular Matrix Controls 13-

(1)

Support Protocol 3: Mechanical Compression of the Fibroblast-Derived Three-Dimensional Matrix 13-

(2)

Support Protocol 4: Solubilization of Fibroblast-Derived Three-Dimensional Matrix 13-

(1)

13.11 Proteoglycan Isolation and Analysis 13-

(8)

Basic Protocol 1: Isolation of Proteoglycans from Cultured Cells 13-

Alternate Protocol: Isolation of Proteoglycan Pools 13-5

(51)

Support Protocol: Radiolabeling of Proteoglycans with 35SO4 or [3H]Glucosamine 13-

(1)

Basic Protocol 2: Anion-Exchange Chromatography Purification of Proteoglycans with DEAF-Sephacel 13-

(1)

Basic Protocol 3: Analysis of Proteoglycans by Size-Exclusion Chromatography 13-

(1)

Basic Protocol 4: Analysis of Glycosaminoglycan Size Following Alkaline Elimination 13-

(1)

Basic Protocol 5: Analysis of Glycosaminoglycan Size Following Papain Digestion 13-

(1)

Basic Protocol 6: Analysis of Glycosaminoglycan Content and Protein Core by GAG Degradation with Lyases 13-

(1)

Basic Protocol 7: Treatment with Nitrous Acid to Degrade Heparan Sulfate 13-

(1)

Basic Protocol 8: Analysis of GAG Type and Core Protein 13-

(1)

Basic Protocol 9: Analysis of GAG Size 13-

(1)

Basic Protocol 10: Immunoprecipitation of Proteoglycans 13-

(1)

13.12 Matrix Metalloproteinases 13-

Basic Protocol 1: Dissolution and Degradation of Collagen Fibrils by Live Cells 13-

(1)

Support Protocol: Preparing Rat Tail Tendon Collagen Type I 13-

(2)

Basic Protocol 2: Gelatin/Casein Zymography 13-

(2)

Basic Protocol 3: Reverse Zymography 13-

14 Cell Motility

14.1 Chemotaxis Assays for Eukaryotic Cells 14-

(9)

Strategic Planning 14-

(1)

Basic Protocol 1: Filter Assay for Chemotaxis 14-

(3)

Support Protocol 1: Calculating the Distance Cells Are Expected to Move in Thick Filters in the Absence of Chemotaxis 14-

(1)

Basic Protocol 2: Under-Agarose Chemotaxis Assay 14-

(1)

Basic Protocol 3: Small Population Chemotaxis Assay 14-

(1)

Basic Protocol 4: Bridge Chemotaxis Assay 14-

(1)

Basic Protocol 5: Pipet Chemotaxis Assay 14-

(1)

14.2 Invasion Assays 14-

(2)

Basic Protocol: Measuring Invasion Through a Matrix 14-

(1)

Support Protocol: Preparation of Matrigel-Coated Filters 14-

(1)

14.3 Cell Traction 14-

(8)

Basic Protocol 1: Measuring Cell Traction on Wrinkling Substrates 14-

(1)

Support Protocol 1: Calibrating Microneedles 14-

(1)

Alternate Protocol 1: Measuring Cell Traction on Wrinkling Substrates with an Alternative Polymer 14-

(1)

Alternate Protocol 2: Measuring Cell Traction on Nonwrinkling Substrates 14-

(1)

Support Protocol 2: Preparing a Modified Airbrush Apparatus 14-

(1)

Basic Protocol 2: Measuring Cell Traction on Micromachined Substrates 14-

(3)

Support Protocol 3: Silanizing Coverslips 14-

(1)

Support Protocol 4: Preparing a Polarized Reflection Cube 14-

(1)

14.4 Cell Wound Assays 14-

(6)

Strategic Planning 14-

(1)

Basic Protocol: Wound Detection in Cultured Monolayers Using Fluorescein Dextran 14-

(1)

Alternate Protocol 1: Wound Detection in Mammalian Tissues Using Fluorescein Dextran 14-

(2)

Alternate Protocol 2: Wound Detection Using Albumin As a Wound Tracer 14-

(2)

14.5 Dictyostelium Cell Dynamics 14-

Basic Protocol 1: Imaging GFP-Labeled Proteins in Live Single Cells 14-

(1)

Alternate Protocol 1: Imaging GFP-Labeled Proteins Following a Uniform Increase in Chemoattractant 14-

(1)

Alternate Protocol 2: Imaging GFP-Labeled Proteins in a Chemoattractant Gradient 14-

(1)

Basic Protocol 2: Imaging GFP-Labeled Proteins in Aggregation Stream and Mound Preparations 14-

(1)

Support Protocol 1: Plasmid Construction and Transformation 14-

(2)

Support Protocol 2: Phenotypic Screening by Development on Non-Nutrient Agar 14-

15 Organelle Motility

15.1 Microtubule/Organelle Motility Assays 15-

(11)

Basic Protocol: MT/Organelle Motility Assays 15-

(3)

Support Protocol 1: Preparation of Simple Perfusion Chambers and Coverslips 15-

(1)

Support Protocol 2: Preparation of Sea Urchin Sperm Axonemes 15-

(2)

Support Protocol 3: Preparation of Porcine Brain Tubulin 15-

(3)

Support Protocol 4: Preparation of Rat Liver Cell Cytosol 15-

(1)

Support Protocol 5: Preparation of Rat Liver Organelle Fractions 15-

(1)

15.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin 15-

(5)

Basic Protocol: Analyzing Actin Translocation by Myosin 15-

(3)

Support Protocol 1: Preparation of Flow Cells 15-

(1)

Support Protocol 2: Purification of Actin 15-

(1)

Support Protocol 3: Preparation of Rhodamine Phalloidin-Labeled Actin 15-

(1)

15.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with Green Fluorescent Protein (GFP) 15-

(2)

Basic Protocol: Transient Expression for Visualization of ER and Golgi Probes in Leaves 15-

(2)

15.4 Movement of Nuclei 15-

Basic Protocol: Nuclear Motility Assay 15-

(2)

Support Protocol 1: Centrosome Isolation from Lymphocytes 15-

(3)

Support Protocol 2: Titration of Concentrated Centrosomes 15-

(1)

Support Protocol 3: Preparing Fractionated Interphase Extracts for Nuclear Assembly 15-

(3)

Support Protocol 4: Preparation of High-Speed Supernatant (HSS) 15-

(1)

Support Protocol 5: Nuclear Assembly Using DNA-Coated Magnetic Beads as Template 15-

Appendices

A1 Reagents and Solutions A1-

(1)

A2 Useful Measurements and Data A2-

(1)

2A Useful Measurements and Data A2-

(2)

2B Compendium of Drugs Commonly Used in Cell Biology Research A2-

(14)

2C Data on Radioisotopes A2-

(3)

2D Absorption and Emission Maxima for Common Fluorophores A2-

(5)

2E Centrifuges and Rotors A2-

A3 Commonly Used Techniques A3-

(1)

3A Spectrophotometric Determination of Protein Concentration A3-

(4)

3B Colorimetric Detection and Quantitation of Total Protein A3-

(14)

3C Dialysis and Concentration of Protein Solutions A3-

(3)

3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy A3-

(4)

3E Enzymatic Amplification of DNA by PCR; Standard Procedures and Optimization A3-

(6)

3F Micro RT-PCR A3-

A4 Selected Suppliers of Reagents and Equipment A4-

References

Index

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Short Protocols in Cell Biology

0471483397

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Table of Contents

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