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Physical and Chemical Methods
Methods in Immunology and Immunochemistry, Vol. 2
- 1st Edition - May 12, 2014
- Editors: Curtis A. Williams, Merrill W. Chase
- Language: English
- Paperback ISBN:9 7 8 - 1 - 4 8 3 2 - 0 9 5 6 - 2
- eBook ISBN:9 7 8 - 1 - 4 8 3 2 - 2 2 9 9 - 8
Methods in Immunology: Volume II, Physical and Chemical Methods is a collection of papers dealing with electrophoresis, analytical ultracentrifugation, dialysis, ultrafiltration,… Read more
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Request a sales quoteMethods in Immunology: Volume II, Physical and Chemical Methods is a collection of papers dealing with electrophoresis, analytical ultracentrifugation, dialysis, ultrafiltration, cellulose ion exchangers, and chromatographic separation of macromolecules on porous gels. Some papers explain the applications of radioisotopes, optical analysis, and chemical analysis of proteins, carbohydrates, lipids, and nucleic acid. One paper describes the theory of electro-migration. Factors such as electrical charge or frictional coefficients govern the rate of migration of charged particles in an electric field. The differences found in their velocities can be used to separate substances or analyze them. Mobility is a characteristic property of molecules and can also be influenced by the composition of the medium or solution. Dialysis separates solvents too large to diffuse through a barrier from smaller solutes; ultrafiltration (reverse osmosis) forces solvent and solutes up to a certain critical size through the barrier by a high pressure on one side. The book notes that the membrane never becomes plugged in dialysis because of some opposite movement of the solvent. Another paper points out that the significance of radioactive tracers in immunochemistry employed to identify and label macromolecules functioning as antigens and antibodies. The collection can prove valuable to bio-chemists, cellular biologists, micro-biologists, developmental biologists, and scientists involved in immunological research.
Contributors to Volume II
Preface
Contents of Other Volumes
Chapter 6. Electrophoresis
A. Factors Governing the Rate of Migration of Charged Particles in an Electric Field
Definition
Electromigration Theory
Electrophoretic Friction
Relaxation Effect
Ionic Strength Effects
Buffer Ion Effects
B. Moving-Boundary Electrophoresis
Historical
Procedures and Concepts
Criteria of Purity
Multiple Moving Boundaries
Demonstrations of Protein-Protein Interactions
Interaction of Proteins with Small Molecules
Fractionation Potentiality
Moving Boundary vs Zone Electrophoresis
C. Analytical Zone Electrophoresis
1. General Survey of Methods and Matrices
2. Cellulose Acetate Membranes (CAM)
Apparatus and Procedure
Fixing and Staining (Proteins, Lipoproteins)
Enzyme Localization
3. Starch Gel Electrophoresis
Theoretical Considerations
Apparatus and Procedure for Vertical Starch Gel
Buffers
Slicing and Staining
Electrophoresis: Common and High-Voltage Separations
Urea-Starch Gel and Urea-Mercaptoethanol-Starch Gel with Acid and Alkaline Buffers
Two-Dimensional Electrophoresis Techniques
4. Disc Electrophoresis, Acrylamide Gel Columns
Theory
Apparatus
Reagents and Solutions
Procedures
5. Acrylamide Gel Electrophoresis
General Consideration
Apparatus
Procedure
Two-Dimensional Gel Electrophoresis
Discontinuous Gel Systems
Handling of Large-Pore Gels
Staining
Recovery of Separated Components
D . Preparative Zone Electrophoresis
1. Zone Electrophoresis on Powder Blocks
General Discussion
Methodology
Choice of Supporting Medium
Applications: Immunoglobulins and Fragments
Ferritin-Globulin Complex
Complement Components
Carbohydrate Separation
2. Column Electrophoresis
General Considerations
Apparatus for Small-Scale Preparations
Anticonvection Materials
Procedure for Serum Fractionation on Sephadex G-25
Large-Scale Preparations
3. Preparative Electrophoresis in Horizontal Gels
General Method of Electrophoresis into Flowing Buffer
Apparatus
Example: Separation of Adenosine Nucleotides
4. Electrophoresis-Convection
Principles
Apparatus and Resolution
Fractionation of y-Globulins and Serum
Other Examples
Chapter 7. Ultracentrifugation
Preparative and Analytical Ultracentrifugation
A. Introduction and Applications
B. Moving-Zone Methods
C. Moving-Zone Methods
D. Isodensity Methods
E. Separation of Cellular Organelles
F. Classical Sedimentation Equilibrium Methods
G. Summary of Mathematical Formulas
Chapter 8 . Dialysis and Ultrafiltration
Dialysis and Ultrafiltration
Definitions
Cellophane Membranes
Analytical Dialysis Cell
Preparative Dialysis
Countercurrent Thin-Layer Dialysis
Ultrafiltration
Methods for Altering the Porosity of Cellophane
Chapter 9 . Chromatographic Separations of Macromolecules on Porous Gels and Cellulose Ion Exchangers
A. Introductions
B. Chromatography on Gels
1. Chromatography on Sephadex
Exclusion Limit of Different Cross-Linked Dextrans
Adsorption Properties
Eluant Choice
Column Dimensions and Buffers
Techniques
2. Chromatography on Polyacrylamide Gels
Preparation of Polyacrylamide Spheres
Packing the Column Techniques
Fractionation of Human Serum
3. Chromatography on Agarose Spheres
Survey
Preparation of Agarose Spheres
Chromatographic Techniques
C. Cellulose Io Exchange Chromatography
Theory and Choice of Ion Exchangers
Preparing the Cellulose Ion Exchangers
Packing and Loading the Column
Elution Principles
Regeneration of Exchangers
D. Analysis of Column Effluents
E. Recovery and Concentration of Proteins
Chapter 10. Optical Analysis
A. Applications of Ultraviolet and Visible Spectroscopy
Absorption Spectra Theory
Molecular Environment and Absorption
Definition of Absorbance, Sources of Absorbance Errors
Specific Antigen-Antibody Reactions: Solutions of Specific Precipitates in Alkali, in Acid, in Detergents
Absorbance of Purified Antibodies
Spectral Changes of Chromophores Due to Protein Binding
B. Turbidimetric Assay Methods: Applications to Antigen-Antibody Reactions
Theoretical Aspects of Turbidimetry
Types of Instruments
Wavelength Selection
Practical Turbidimetry
Chapter 11. Radioisotopes and their Applications
A. Radiolabeling of Macromolecules
External and Internal Labels
Radioactive Isotopes
B. Principles in Choice of Radioactive Traces
Half-Life of Radiation
Radioactive Decay
Specific Radioactivity
Stability of Label in vivo
Biological Half-Life vs Effective Half-Life
C. Laboratory Facilities and Procedures
Considerations of Surfaces, Hoods, Sinks
Care of Glassware and Pipets
General Precautions
Isotopes and Experimental Animals
D. Equipment
Instruments for Counting
Instrument Selection
E. Preparation of Samples
The End-Window Geiger-Mueller Counter: Techniques and Examples
The Liquid Scintillation Counter: Techniques and Examples
F. Evaluation of Counting Data
Correction for Decay
Specific Radioactivity
Statistical Evaluation of Counting Data
Source Materials
G. Autoradiography in Immunological Investigation
Isotope Selection
Histological Preparations
Controls Applying the Photographic Emulsion: Sandwich Technique
Stripping Film
Liquid Emulsions
Exposure and Processing
Histological Staining
Photographic Records
Evaluation of Radioautographs
H. Glossary of Radioisotopes Terminology
Chapter 12. Chemical Analysis
A. Protein Analysis
General Considerations
Moisture and Ash Content
Nitrogen Determination: Kjeldahl Method
Nessler Reaction
Micro-Dumas Method
Determination of Protein: Biuret Method
Ninhydrin Method
For Intact Proteins
For Protein Hydrolysates
Ultraviolet Spectroscopy for Protein Determination
Monitoring Chromatographic Effluents
Nucleic Acid and Protein Content of Crude Solutions
B. Carbohydrates Analysis
Reducing Sugars on Hydrolysis
General Carbohydrate Reactions
Total Hexoses
Keto Sugars
Pentoses
Methylpentoses
Hexosamine and N-Acetylhexosamines
Uronic Acid
Sialic Acids
C. Preparation of Lipoprotiens and Analysis of Lipids
Lipoprotein Classes
Ultracentrifugal Separation
Analysis of Serum Lipids
Cholesterol
Triglycerides
Free Fatty Acids
Lipid Phosphorus
Extraction and Fractionation of Lipids
Thin-Layer Chromatography
D. Nucleic Acid Analysis
1. Methods for Characterizing Deoxyribonucleic Acid Immune Systems
Extraction and Purification of DNA
Base Ratio Analysis
Diphenylamine Reaction
Determination of Chain Length
Immunochemical Methods for Determining Conformation of the Antigen: Denaturation and Renaturation
Methods for Identification of the Antigenic Determinants: Removal of Purine Residues
Removal of Pyrimidine Residues
Removal of Guanine Residues
2. Isolation and Characterization of Ribonucleic Acid
Extraction and Purification of RNA
Isolation of RNA from Subcellular Components
Spectrophotometric Analysis
Orcinol Reaction for Riboses
Contamination by DNA and Proteins
Characterization of RNA
Size
Nucleotide Composition and Separation
Measurement of Template Activity
Appendix I
Spectral Properties of Proteins and Small Molecules of Immunological Interest
Proteins
Immunoglobulins and Component Structures
Purified Antibodies and Component Structures
Diazo Compounds
Nitrophenyl, Dinitrophenyl Compounds
Nucleic Acid Components
Penicillins
Trinitrophenyl Compounds
Naphthalene Derivatives
Iodinated Pyridine
Fluorescein
Lissamine Rhodamine
Organic Solvents
Iodinated Tyrosines, et cetera
Appendix II
Buffers
Buffering Capacity and Formulations
Volatile Buffers
Microbial Growth
Absolute Reference Standard Buffers
Glass Electrode Calibration
Buffer Components
Buffer Formulas Indicator Dyes: Indicator Papers, Indicator Solutions
Isotonic Concentrations of Common Chemicals
Appendix III
Power Block Electrophoresis
Author Index
Subject Index
- No. of pages: 480
- Language: English
- Edition: 1
- Published: May 12, 2014
- Imprint: Academic Press
- Paperback ISBN: 9781483209562
- eBook ISBN: 9781483222998