- Chapter 1: Antibody Production by the Immune System
- Chapter 2: The Antibody Molecule
- Chapter 3: Antibody-Antigen Interactions
- Chapter 4: Antibody Responses
- Chapter 5: Selecting the Antigen
- Protocol 1: Modifying Antigens by Dinitrophenol Coupling
- Protocol 2: Modifying Antigens by Arsynyl Coupling
- Protocol 3: Modifying Protein Antigens by Denaturation
- Protocol 4: Preparing Immune Complexes for Injection
- Protocol 5: Coupling Antigens to Red Blood Cells
- Protocol 6: Coomassie Brilliant Blue Staining
- Protocol 7: Sodium Acetate Staining
- Protocol 8: Copper Chloride Staining
- Protocol 9: Side-Strip Method
- Protocol 10: Fragmenting a Wet Gel Slice
- Protocol 11: Lyophilization of a Gel Slice
- Protocol 12: Electroelution of Protein Antigens from Polyacrylamide Gel Slices
- Protocol 13: Electrophoretic Transfer to Nitrocellulose Membranes
- Protocol 14: Autoradiography
- Protocol 15: Cross-Linking Peptides to KLH with Maleimide
- Protocol 16: Preparing GST-Fusion Proteins from Bacteria
- Protocol 17: Preparing His-Fusion Proteins from Bacteria
- Protocol 18: Preparing MBP-Fusion Proteins from Bacteria
- Protocol 19: Sarkosyl Preparation of Antigens from Bacterial Inclusion Bodies
- Protocol 20: Preparing mFc- and hFc-Fusion Proteins from Mammalian Cells
- Protocol 21: Preparing Live Cells for Immunization
- Protocol 22: Preparing Antigens Using a Baculovirus Expression System
- Chapter 6: Immunizing Animals
- Protocol 1: Administering Anesthesia to Mice, Rats, and Hamsters
- Protocol 2: Administering Anesthesia to Rabbits
- Protocol 3: Preparing Freund's Adjuvant
- Protocol 4: Using Ribi Adjuvant
- Protocol 5: Using Hunter's TiterMax Adjuvant
- Protocol 6: Using Magic Mouse Adjuvant
- Protocol 7: Preparing Aluminum Hydroxide (Alum) Adjuvant
- Protocol 8: Injecting Rabbits Subcutaneously
- Protocol 9: Subcutaneous Injections with Adjuvant into Mice and Rats
- Protocol 10: Subcutaneous Injections without Adjuvant into Mice and Rats
- Protocol 11: Immunizing Mice and Rats with Nitrocellulose-Bound Antigen
- Protocol 12: Injecting Rabbits Intramuscularly
- Protocol 13: Injecting Rabbits Intradermally
- Protocol 14: Injecting Rabbits Intravenously
- Protocol 15: Injecting Mice Intravenously
- Protocol 16: Intraperitoneal Injections with Adjuvant into Mice and Rats
- Protocol 17: Intraperitoneal Injections without Adjuvant into Mice and Rats
- Protocol 18: Immunizing Mice, Rats, and Hamsters in the Footpad or Hock
- Protocol 19: Sampling Rabbit Serum from the Marginal Ear Vein
- Protocol 20: Sampling Mouse Serum from the Tail Vein
- Protocol 21: Sampling Rat Serum from the Tail Vein
- Protocol 22: Sampling Mouse and Rat Serum from the Retro-Orbital Sinus
- Protocol 23: Sampling Mouse and Rat Serum from the Submandibular Vein
- Protocol 24: Sampling Mouse and Rat Serum from the Saphenous Vein
- Protocol 25: Serum Preparation
- Protocol 26: Induction of Ascites Using Freund's Adjuvant
- Protocol 27: Induction of Ascites in BALB/c Mice Using Myeloma Cells
- Protocol 28: Standard Immunization of Mice, Rats, and Hamsters
- Protocol 29: Standard Immunization of Rabbits
- Protocol 30: Repetitive Immunization at Multiple Sites (RIMMS) of Mice, Rats, and Hamsters
- Protocol 31: Subtractive Immunization for Mice, Rats, and Hamsters
- Protocol 32: Decoy Immunization for Mice, Rats, and Hamsters
- Protocol 33: Adoptive Transfer Immunization of Mice
- Protocol 34: cDNA Immunization of Mice, Rats, and Hamsters
- Protocol 35: Euthanizing Mice, Rats, and Hamsters Using CO2 Asphyxiation
- Protocol 36: Harvesting Spleens from Mice, Rats, and Hamsters
- Chapter 7: Generating Monoclonal Antibodies
- Protocol 1: Antibody Capture in Polyvinyl Chloride Wells: Enzyme-Linked Detection (Indirect ELISA)
- Protocol 2: Antibody Capture in Polyvinyl Chloride Wells: Enzyme-Linked Detection When Immunogen Is an Immunoglobulin Fusion Protein (Indirect ELISA to Detect Ig Fusion Proteins)
- Protocol 3: Antibody Capture on Nitrocellulose Membrane: Dot Blot
- Protocol 4: Antibody Capture on Nitrocellulose Membrane: High-Throughput Western Blot Assay for Hybridoma Screening
- Protocol 5: Antibody Capture on Whole Cells: Cell-Surface Binding (Surface Staining by Flow Cytometry/FACS)
- Protocol 6: Antibody Capture on Permeabilized Whole Cells Binding (Intracellular Staining by Flow Cytometry/FACS)
- Protocol 7: Antibody Capture on Whole Cells: Cell-Surface Binding (Surface Staining by Immunofluorescence)
- Protocol 8: Antibody Capture on Permeabilized Whole Cells (Immunofluorescence)
- Protocol 9: Antibody Capture on Tissue Sections (Immunohistochemistry)
- Protocol 10: Antigen Capture in 96-Well Plates (Capture or Sandwich ELISA)
- Protocol 11: Antigen Capture on Nitrocellulose Membrane: Reverse Dot Blot
- Protocol 12: Antigen Capture in Solution: Immunoprecipitation
- Protocol 13: Screening for Good Batches of Fetal Bovine Serum
- Protocol 14: Preparing Peritoneal Macrophage Feeder Plates
- Protocol 15: Preparing Myeloma Cell Feeder Layer Plates
- Protocol 16: Preparing Splenocyte Feeder Cell Cultures
- Protocol 17: Preparing Fibroblast Feeder Cell Cultures
- Protocol 18: Screening for Good Batches of Polyethylene Glycol
- Protocol 19: Polyethylene Glycol Fusion
- Protocol 20: Fusion by Sendai Virus
- Protocol 21: Electro Cell Fusion
- Protocol 22: Single-Cell Cloning by Limiting Dilution
- Protocol 23: Single-Cell Cloning by Growth in Soft Agar
- Protocol 24: Determining the Class and Subclass of a Monoclonal Antibody by Ouchterlony Double-Diffusion Assays
- Protocol 25: Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Antigen-Coated Plates
- Protocol 26: Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Anti-Immunoglobulin Antibody-Coated Plates
- Chapter 8: Growing Hybridomas
- Protocol 1: Counting Myeloma or Hybridoma Cells
- Protocol 2: Viability Checks
- Protocol 3: Freezing Cells for Liquid Nitrogen Storage
- Protocol 4: Recovering Cells from Liquid Nitrogen Storage
- Protocol 5: Ridding Cell Lines of Contaminating Microorganisms by Antibiotics
- Protocol 6: Ridding Cell Lines of Contaminating Microorganisms with Peritoneal Macrophages
- Protocol 7: Ridding Cell Lines of Contaminating Microorganisms by Passage through Mice
- Protocol 8: Testing for Mycoplasma Contamination by Growth on Microbial Medium
- Protocol 9: Testing for Mycoplasma Contamination by Hoechst Dye 33258 Staining
- Protocol 10: Testing for Mycoplasma Contamination Using PCR
- Protocol 11: Testing for Mycoplasma Contamination Using Reporter Cells
- Protocol 12: Ridding Cells of Mycoplasma Contamination Using Antibiotics and Single-Cell Cloning
- Protocol 13: Ridding Cells of Mycoplasma by Passage through Mice
- Protocol 14: Inducing and Collecting Ascites
- Protocol 15: Collecting Tissue Culture Supernatants
- Protocol 16: Storing Tissue Culture Supernatants and Ascites
- Protocol 17: Selecting Myeloma Cells for HGPRT Mutants with 8-Azaguanine
- Chapter 9: Characterizing Antibodies
- Chapter 10: Antibody Purification and Storage
- Chapter 11: Engineering Antibodies
- Chapter 12: Labeling Antibodies
- Protocol 1: Labeling Antibodies with NHS-LC-Biotin
- Protocol 2: Biotinylating Antibodies Using Biotin Polyethylene Oxide (PEO) Iodoacetamide
- Protocol 3: Biotinylating Antibodies Using Biotin-LC Hydrazide
- Protocol 4: Labeling Antibodies Using NHS-Fluorescein
- Protocol 5: Labeling Antibodies Using a Maleimido Dye
- Protocol 6: Conjugation of Antibodies to Horseradish Peroxidase
- Protocol 7: Labeling Antibodies with Cy5-Phycoerythrin
- Protocol 8: Labeling Antibodies Using Europium
- Protocol 9: Labeling Antibodies Using Colloidal Gold
- Protocol 10: Iodination of Antibodies with Immobilized Iodogen
- Chapter 13: Immunoblotting
- Protocol 1: Preparing Whole-Cell Lysates for Immunoblotting
- Protocol 2: Preparing Protein Solutions for Immunoblotting
- Protocol 3: Preparing Immunoprecipitations for Immunoblotting
- Protocol 4: Resolving Proteins for Immunoblotting by Gel Electrophoresis
- Protocol 5: Semi-Dry Electrophoretic Transfer
- Protocol 6: Wet Electrophoretic Transfer
- Protocol 7: Staining the Blot for Total Protein with Ponceau S
- Protocol 8: Blocking and Incubation with Antibodies: Immunoblots Prepared with Whole-Cell Lysates and Purified Proteins (Straight Western Blotting)
- Protocol 9: Blocking and Incubation with Antibodies of Immunoblots Prepared with Immunoprecipitated Protein Antigens (Immunoprecipitation/Western Blotting)
- Protocol 10: Detection with Enzyme-Labeled Reagents
- Protocol 11: Detection with Fluorochromes
- Chapter 14: Immunoprecipiation
- Protocol 1: Metabolic Labeling of Antigens with [35S]Methionine
- Protocol 2: Pulse-Chase Labeling of Antigens with [35S]Methionine
- Protocol 3: Metabolic Labeling of Antigens with [32P]Orthophosphate
- Protocol 4: Freezing Cell Pellets for Large-Scale Immunoprecipitation
- Protocol 5: Detergent Lysis of Tissue Culture Cells
- Protocol 6: Detergent Lysis of Animal Tissues
- Protocol 7: Lysis Using Dounce Homogenization
- Protocol 8: Differential Detergent Lysis of Cellular Fractions
- Protocol 9: Lysing Yeast Cells with Glass Beads
- Protocol 10: Lysing Yeast Cells Using a Coffee Grinder
- Protocol 11: Denaturing Lysis
- Protocol 12: Cross-Linking Antibodies to Beads Using Dimethyl Pimelimidate (DMP)
- Protocol 13: Cross-Linking Antibodies to Beads with Disuccinimidyl Suberate (DSS)
- Protocol 14: Immunoprecipitation
- Protocol 15: Tandem Immunoaffinity Purification Using Anti-FLAG and Anti-HA Antibodies
- Protocol 16: Chromatin Immunoprecipitation
- Chapter 15: Immunoassays
- Chapter 16: Cell Staining
- Protocol 1: Growing Adherent Cells on Coverslips or Multiwell Slides
- Protocol 2: Growing Adherent Cells on Tissue Culture Dishes
- Protocol 3: Attaching Suspension Cells to Slides Using the Cytocentrifuge
- Protocol 4: Attaching Suspension Cells to Slides Using Poly-L-Lysine
- Protocol 5: Preparing Cell Smears
- Protocol 6: Attaching Yeast Cells to Slides Using Poly-L-Lysine
- Protocol 7: Preparing Frozen Tissue Sections
- Protocol 8: Preparing Paraffin Tissue Sections
- Protocol 9: Additional Protocol: Heat-Induced Epitope Retrieval
- Protocol 10: Preparing Cell Smears from Tissue Samples or Cell Cultures
- Protocol 11: Embedding Cultured Cells in Matrigel
- Protocol 12: Fixing Attached Cells in Organic Solvents
- Protocol 13: Fixing Attached Cells in Paraformaldehyde or Glutaraldehyde
- Protocol 14: Fixing Suspension Cells with Paraformaldehyde
- Protocol 15: Lysing Yeast
- Protocol 16: Binding Antibodies to Attached Cells or Tissues
- Protocol 17: Binding Antibodies to Cells in Suspension
- Protocol 18: Detecting Horseradish PeroxidaseLabeled Cells Using Diaminobenzidine
- Protocol 19: Detecting Horseradish PeroxidaseLabeled Cells Using Diaminobenzidine and Metal Salts
- Protocol 20: Detecting Horseradish PeroxidaseLabeled Cells Using Chloronaphthol
- Protocol 21: Detecting Horseradish PeroxidaseLabeled Cells Using Aminoethylcarbazole
- Protocol 22: Detecting Alkaline PhosphataseLabeled Cells Using NABP-NF
- Protocol 23: Detecting Alkaline PhosphataseLabeled Cells Using BCIP-NBT
- Protocol 24: Detecting -Galactosidase-Labeled Cells Using X-Gal
- Protocol 25: Detecting Fluorochrome-Labeled Reagents
- Protocol 26: Detecting Gold-Labeled Reagents
- Protocol 27: Detecting Iodine-Labeled Reagents
- Protocol 28: Counterstaining Cells
- Protocol 29: Mounting Cell or Tissue Samples in DPX
- Protocol 30: Mounting Cell or Tissue Samples in Gelvatol or Mowiol
- Chapter 17: Antibody Screening using High Throughput Flow Cytometry
- Chapter 18: Appendix I: Electrophoresis
- Chapter 19: Appendix II: Protein Techniques
- Chapter 20: Appendix III: General Information
- Chapter 21: Appendix IV: Bacterial Expression
- Chapter 22: Appendix V: Cautions
- Chapter 23: Index
Metabolic Labeling of Antigens with [32P]Orthophosphate
(Protocol summary only for purposes of this preview site)Metabolic labeling incorporating [32P]orthophosphate, available in a wide range of specific activities, is a highly sensitive method for determining whether proteins are phosphorylated or not (Bond et al. 1999; Bursik et al. 1999; Macnaughton and Lai 2002; Matsushita et al. 2002; Girao and Pereira 2003; Garbarino-Pico et al. 2004; He et al. 2004; Smal et al. 2006). Cells typically have a large requirement for phosphate and when cultured in the presence of [32P]orthophosphate will readily take up and incorporate the radiolabeled orthophosphate used for the phosphorylation of proteins.
