Density Gradient Centrifugation

Editors’ preface

Part I R. Hinton and M. Dobrota Density Gradient Centrifugation

Density Gradient Centrifugation

Chapter 1: Introduction to zonal centrifugation

1.1. The first applications of centrifugation in biology

1.2. Centrifugal techniques

1.3. The development of centrifuges and rotors

1.4. Uses and limitations of centrifugal techniques

1.5. Design of a centrifuge laboratory

1.6. Safety of centrifuges

1.7. Care of rotors

1.8. Guarantees on rotors

Chapter 2: Theoretical aspects of centrifugal separations

2.1. Theory of rate-zonal separations

2.2. Theory of isopycnic banding

2.3. Effects of density gradient solutes on subcellular structures

Chapter 3: Conditions for a centrifugal separation

3.1. Choice of approach

3.2. Choice of rotor

3.3. Density gradient solutes

3.4. Choice of gradient

Chapter 4: Centrifugation in conventional rotors

4.1. Rate-zonal centrifugation

4.2. Isopycnic zonal centrifugation

Chapter 5: Centrifugation in zonal rotors

5.2 Conventional, non-reorienting zonal rotors

5.2 Reorienting zonal rotors

Chapter 6: Assay of fractions separated by density gradient centrifugation

6.1. Enzyme and chemical assays on fractions

6.2. Electron microscopic examination of fractions

6.3. Assessment of results from density gradient separations

6.4. Calculation of sedimentation coefficients

Chapter 7: Applications of density gradient centrifugation

7.1. Separation of living cells

7.2. Separation of cell organelles from mammalian tissues

7.3. Separation of subcellular structures from plant cells

7.4. Separation of subcellular components from unicellular organisms

7.5. Fractionation of macromolecules

7.6. Other applications of density gradient centrifugation in biochemistry

7.7. Other applications of density gradient centrifugation

Chapter 8: Artefacts arising during centrifugal separations

8.1. Damage to particles during centrifugation

8.2. Factors affecting the accuracy of assays performed on fractions from density gradients

8.3. Uncertainties in estimates of particle density and sedimentation coefficient

Chapter 9: Future prospects for density gradient centrifugation

9.1. Centrifuge design

9.2. Developments in centrifuge rotors

9.3. Developments in ancillary systems

9.4. Uses of centrifugal methods

Note added in proof

Acknowledgements

Appendices

Appendix I

Appendix II

Appendix III

Appendix IV

References

Subject index

Part II T. Chard An Introduction to Radioimmunoassay and Related Techniques

An Introduction to Radioimmunoassay and Related Techniques

List of abbreviations

Chapter 1: The background to radioimmunoassay

1.1. Introduction

1.2. Terminology

1.3. Early development of radioimmunoassay

1.4. Basic principles of binding assays

1.5. Binder dilution curves and standard curves

1.6. Methods of plotting the standard curve

1.7. The importance of K value

1.8. The measurement of K value

1.9. A model system for binding assays

1.10. Some implications of the model system

Chapter 2: Requirements for a binding assay – purified ligand

2.1. Requirements for a binding assay

2.2. The need for purified ligand

2.3. Availability of pure ligand

2.4. Dissimilarity between purified ligand and endogenous ligand

2.5. Standards

2.6. Storage of materials

Chapter 3: Requirements for binding assays – tracer ligand

3.1. Radioactive isotopes

3.2. Counting of radioactive isotopes

3.3. Choice of a counter

3.4. Some practical aspects of isotope counting

3.5. Essential characteristics of a tracer

3.6. Preparation of tracers

3.7. Iodinated tracers

3.8. Alternative labels for tracers

Chapter 4: Requirements for a binding assay – the binder

4.1. Characteristics required of a binder

4.2. Antibodies

4.3. Cell receptors

4.4. Circulating binding proteins

4.5. Radioassay for the detection of endogenous antibodies and circulating binding proteins

Chapter 5: Requirements for a binding assay – separation of bound and free ligand

5.1. Efficiency of separation methods

5.2. Practicality of separation methods

5.3. Methods for the separation of bound and free ligand

5.4. Immunoradiometric techniques

Chapter 6: Requirements for a binding assay – extraction of ligand from biological fluids

6.1. Extraction for concentration of ligand

6.2. Extraction for purification of ligand

6.3. General aspects of extraction procedures

Chapter 7: Requirements for binding assays – calculation of results

7.1. Calculation of results by simple manual extrapolation

7.2. Linearisation of the standard curve

7.3. Electronic aids to calculation of results

7.4. Estimation of confidence limits to the results

Chapter 8: Characteristics of binding assays – sensitivity

8.1. Definition of sensitivity

8.2. Methods of increasing the sensitivity of a binding assay

8.3. Methods of decreasing the sensitivity of an assay

8.4. Targeting of binding assay – the importance of ranges

8.5. Optimisation of an assay by theoretical analysis

8.6. Conclusions

Chapter 9: Characteristics of binding assays – specificity

9.1. Definition of specificity

9.2. Specific non-specificity

9.3. Non-specific non-specificity

Chapter 10: Characteristics of binding assays – precision

10.1. Definitions

10.2. Factors affecting precision

10.3. Methods for monitoring the precision of a binding assay

10.4. Methods for optimising the precision of a binding assay

Chapter 11: Characteristics of binding assays – relation to other types of assay

11.1. Definition

11.2. Receptor assays

11.3. Assays using circulating binding proteins

11.4. Immunoassays

11.5. Conclusions

Chapter 12: Automation of binding assays

12.1. General

12.2. Identification and dispensing of the sample

12.3. Addition of reagents

12.4. Incubation

12.5. Separation of bound and free ligand

12.6. Counting of radioactivity

12.7. Calculation of results

12.8. Conclusions

Chapter 13: Organisation of assay services

13.1. Who should perform radioimmunoassay?

13.2. Organisation of an assay laboratory

13.3. Organisation of assay services

Appendices

Appendix I

Appendix II

Appendix III

Appendix IV

Appendix V

References

Subject index