Physical and Chemical Methods

Physical and Chemical Methods

Methods in Immunology and Immunochemistry, Vol. 2

1st Edition - January 1, 1968

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  • Editors: Curtis A. Williams, Merrill W. Chase
  • eBook ISBN: 9781483222998

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Methods 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.

Table of Contents

  • Contributors to Volume II


    Contents of Other Volumes

    Chapter 6. Electrophoresis

    A. Factors Governing the Rate of Migration of Charged Particles in an Electric Field


    Electromigration Theory

    Electrophoretic Friction

    Relaxation Effect

    Ionic Strength Effects

    Buffer Ion Effects

    B. Moving-Boundary Electrophoresis


    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


    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



    Reagents and Solutions


    5. Acrylamide Gel Electrophoresis

    General Consideration



    Two-Dimensional Gel Electrophoresis

    Discontinuous Gel Systems

    Handling of Large-Pore Gels


    Recovery of Separated Components

    D . Preparative Zone Electrophoresis

    1. Zone Electrophoresis on Powder Blocks

    General Discussion


    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


    Example: Separation of Adenosine Nucleotides

    4. Electrophoresis-Convection


    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


    Cellophane Membranes

    Analytical Dialysis Cell

    Preparative Dialysis

    Countercurrent Thin-Layer Dialysis


    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


    2. Chromatography on Polyacrylamide Gels

    Preparation of Polyacrylamide Spheres

    Packing the Column Techniques

    Fractionation of Human Serum

    3. Chromatography on Agarose Spheres


    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



    Hexosamine and N-Acetylhexosamines

    Uronic Acid

    Sialic Acids

    C. Preparation of Lipoprotiens and Analysis of Lipids

    Lipoprotein Classes

    Ultracentrifugal Separation

    Analysis of Serum Lipids



    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


    Nucleotide Composition and Separation

    Measurement of Template Activity

    Appendix I

    Spectral Properties of Proteins and Small Molecules of Immunological Interest


    Immunoglobulins and Component Structures

    Purified Antibodies and Component Structures

    Diazo Compounds

    Nitrophenyl, Dinitrophenyl Compounds

    Nucleic Acid Components


    Trinitrophenyl Compounds

    Naphthalene Derivatives

    Iodinated Pyridine


    Lissamine Rhodamine

    Organic Solvents

    Iodinated Tyrosines, et cetera

    Appendix II


    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

Product details

  • No. of pages: 480
  • Language: English
  • Copyright: © Academic Press 1968
  • Published: January 1, 1968
  • Imprint: Academic Press
  • eBook ISBN: 9781483222998

About the Editors

Curtis A. Williams

Merrill W. Chase

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