Physical and Chemical Methods - 1st Edition - ISBN: 9781483197968, 9781483222998

Physical and Chemical Methods

1st Edition

Methods in Immunology and Immunochemistry, Vol. 2

Editors: Curtis A. Williams Merrill W. Chase
eBook ISBN: 9781483222998
Imprint: Academic Press
Published Date: 1st January 1968
Page Count: 480
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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


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© Academic Press 1968
Academic Press
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About the Editor

Curtis A. Williams

Merrill W. Chase

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