Birnie / Rickwood Centrifugal Separations in Molecular and Cell Biology

1. Auflage 2014
ISBN: 978-1-4832-7841-4
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 340 Seiten

Centrifugal Separations in Molecular and Cell Biology focuses on the application of modern centrifugation technology in molecular and cell biology, including the separation and fractionation of biological particles by centrifugation on the preparative and analytical scales. The selection first covers the principles and practices of centrifugation and the bases of centrifugal separations. Discussions focus on the basic concepts of sedimentation theory, centrifugation methods, designing centrifugation experiments, care of centrifuges and rotors, and statistical estimation of molecular parameters. The book also ponders on the practical aspects of rate-zonal centrifugation, including gradient materials, density and viscosity of glycerol solutions, and resolution and gradient shape. The publication examines fractionations in zonal rotors and the quantitative aspects of rate-zonal centrifugation. The text then reviews isopycnic centrifugation in ionic media and analytical centrifugation. Topics include separation by isopycnic banding, large-scale preparative procedures, and density-gradient solutes. The selection is a valuable reference for readers interested in centrifugation technology.

Autoren/Hrsg.

Birnie, G. D.

Rickwood, D.

Inhaltsverzeichnis

1;Front Cover;1
2;Centrifugal Separations in Molecular and Cell Biology;4
3;Copyright Page;5
4;Table of Contents;10
5;Preface;6
6;Contributors;8
7;Chapter 1. Introduction: Principles and Practices of Centrifugation;14
7.1;BASIC CONCEPTS OF SEDIMENTATION THEORY;14
7.2;CENTRIFUGATION METHODS;15
7.3;DESIGNING CENTRIFUGATION EXPERIMENTS;18
7.4;CARE OF CENTRIFUGES AND ROTORS;19
8;Chapter 2. The Bases of
Centrifugal Separations;20
8.1;COMPARISON OF ZONAL CENTRIFUGATION WITH COMPLEMENTARY
TECHNIQUES;20
8.2;THEORY OF RATE-ZONAL EXPERIMENTS;24
8.3;ISOPYCNIC ZONAL EXPERIMENTS;29
8.4;STATISTICAL ESTIMATION OF MOLECULAR PARAMETERS;31
8.5;SIMULATION OF ZONAL EXPERIMENTS;34
8.6;APPENDIX;35
8.7;REFERENCES;43
9;Chapter 3. Practical Aspects of Rate-Zonal Centrifugation;46
9.1;EXPERIMENTAL DESIGN;46
9.2;GRADIENT MATERIALS;48
9.3;RESOLUTION AND GRADIENT SHAPE;53
9.4;LOADING, RUNNING AND ANALYSING GRADIENTS;60
9.5;APPENDIX I: DENSITY AND VISCOSITY OF SUCROSE SOLUTIONS;69
9.6;APPENDIX II: DENSITY AND VISCOSITY OF GLYCEROL SOLUTIONS;71
9.7;REFERENCES;72
10;Chapter 4. Fractionations in
Zonal Rotors;76
10.1;DESIGN AND OPERATION OF BATCH-TYPE ROTORS;77
10.2;FRACTIONATIONS IN BATCH-TYPE
ROTORS;89
10.3;CONTINUOUS-FLOW ZONAL CENTRIFUGATION;113
10.4;CENTRIFUGAL ELUTRIATION;120
10.5;ACKNOWLEDCEMENTS;122
10.6;REFERENCES;122
11;Chapter 5. Rate-Zonal Centrifugation:
Quantitative Aspects;128
11.1;SOME IMPORTANT UNDERLYING ASSUMPTIONS;128
11.2;GUIDE FOR THE COMPUTERIZED CALCULATION OF SEDIMENTATION
COEFFICIENTS;130
11.3;GUIDE FOR THE MANUAL CALCULATION OF SEDIMENTATION
COEFFICIENTS;134
11.4;ACCURACY OF SEDIMENTATION COEFFICIENTS CALCULATED FROM
DENSITY-GRADIENT DATA USING LARGE-SCALE ZONAL ROTORS;138
11.5;OTHER ANALYTICAL USES;146
11.6;APPENDIX I: FORTRAN PROGRAM FOR CALCULATING SEDIMENTATION COEFFICIENTS;148
11.7;APPENDIX II: CALCULATING RADIUS-VOLUME RELATIONSHIPS;151
11.8;APPENDIX III: SEDIM VALUES FOR VARIOUS PARTICLE DENSITIES;152
11.9;APPENDIX IV: CALCULATING THE NATURAL LOGARITHM OF THE
ROTOR RADIUS;176
11.10;REFERENCES;179
12;Chapter 6. Isopycnic Centrifugation in ionic
Media;182
12.1;BASIC PROCEDURES;183
12.2;OPTIMUM DESIGN OF BUOYANT DENSITY-GRADIENT SEPARATIONS;186
12.3;DATA FROM ISOPYCNIC GRADIENTS;209
12.4;SEPARATIONS BY ISOPYCNIC BANDING;212
12.5;LARGE-SCALE PREPARATIVE PROCEDURES;222
12.6;ACKNOWLEDGEMENTS;228
12.7;REFERENCES;228
13;Chapter 7. Isopycnic Centrifugation
in Non-ionic Media;232
13.1;GENERAL TECHNIQUES;232
13.2;DENSITY-GRADIENT SOLUTES;235
13.3;ACKNOWLEDGEMENTS;260
13.4;REFERENCES;260
14;Chapter 8. Analytical Ultracentrifugation;264
14.1;HISTORY AND SCOPE;264
14.2;INSTRUMENTATION;265
14.3;SEDIMENTATION EQUILIBRIUM
ANALYSIS;270
14.4;SEDIMENTATION VELOCITY ANALYSIS;283
14.5;CONCLUSIONS;296
14.6;REFERENCES;297
15;Chapter 9. Characteristics of Ultracentrifuge Rotors
and Tubes;302
15.1;DESIGNS AND TYPES OF ROTOR;302
15.2;CENTRIFUGE TUBES AND CAPS;307
15.3;DERATING ROTORS;313
15.4;ROUTINE MAINTENANCE OF ROTORS;315
15.5;ACKNOWLEDGEMENTS;315
15.6;APPENDIX: PHYSICAL DIMENSIONS AND CHARACTERISTICS OF
ROTORS;316
16;Index;330

Leseproben

1

Introduction: Principles and Practices of Centrifugation

D. RICKWOOD,     Department of Biology, The University of Essex

G.D. BIRNIE,     The Beatson Institute for Cancer Research, Wolfson Laboratory for Molecular Pathology, Glasgow

Publisher Summary

This chapter describes the principles and practices of centrifugation techniques. There are three main types of centrifugal fractionation, namely, (1) differential pelleting (differential centrifugation); (2) rate-zonal density-gradient sedimentation; and (3) isopycnic density-gradient sedimentation. Of these techniques, differential pelleting is the method most commonly used for fractionating material according to size. In this method, the material to be fractionated is initially distributed uniformly throughout the sample solution, which is the sole occupant of the centrifuge tube. The efficiency of the fractionation of particles according to size and shape can be improved markedly by using rate-zonal centrifugation through a density gradient, although this does mean that the sample capacity of each tube is greatly reduced. This technique involves layering the sample on to the top of a liquid column that is stabilized by a gradient of an inert solute, commonly sucrose. The third method for separating particles is isopycnic sedimentation in a gradient whose maximum density exceeds that of the particles. This is an equilibrium technique in which particles are separated on the basis of their buoyant densities, independently of the time of centrifugation and of the size and shape of the particles, although these parameters do determine the rate at which equilibrium is reached and the width of the bands formed at equilibrium.

This book is designed both as a complete guide for the novice and as an aid to the worker who has already had some experience of centrifugal techniques. This introductory chapter discusses the various techniques which are available to the researcher and describes the layout of the book, thus directing the reader to the relevant chapters which provide the information necessary to carry out each type of fractionation.

BASIC CONCEPTS OF SEDIMENTATION THEORY

Essentially, a centrifuge is a device for separating particles from a solution. In biology, the particles are usually cells, subcellular organelles, or large molecules – all of which are called ‘particles’ to simplify the terminology. The physical parameters which determine the extent of fractionation apply equally to such diverse particles as macromolecules and cells, although the nature of the particles (for example, their lability, sensitivity to osmotic pressure, etc.) may place restraints on the centrifugation conditions that can be used. The following is a simplified introduction to some of the basic parameters which govern the sedimentation and separation of particles in a centrifugal field. A detailed analysis of the theoretical aspects of centrifugal separations is given in Chapter 2.

Some of the basic principles of the sedimentation theory originate from Stokes’s law. If the sedimentation of a sphere in a gravitational field is considered it can be shown that, as the velocity of a spherical particle reaches a constant value, the net force on the particle is equal to the force resisting its motion through the liquid. This resisting force is called frictional or drag force. From Stokes’s law it can be calculated that the sedimentation rate, , of a particle is given by

=d2(?p-?m)18µ×g

From this equation, it can be seen that:

1. The sedimentation rate of a given particle is proportional to the square of the diameter, , of the particle.

2. The sedimentation rate is proportional to the difference between the density of the particle and the density of the liquid medium, (?p - ?m).

3. The sedimentation rate is zero when the density of the particle is equal to the density of the liquid medium.

4. The sedimentation rate decreases as the viscosity, µ, of the liquid medium increases.

5. The sedimentation rate increases as the force field, , increases.

The force field relative to the earth’s gravitational field (RCF) exerted during centrifugation is defined by the equation

=?2r980

where is the distance between the particle and the centre of rotation in cm; the rotor speed ? in rad/sec can be calculated from the equation

=rev/min×2p60=rev/min×0.104 72

The sedimentation velocity per unit of centrifugal force is called the sedimentation coefficient, :

=1?2r×drdt

where d/d is the rate of movement of the particle in cm/sec. Sedimentation coefficients are usually expressed in svedbergs (S), equivalent to 10-13 sec. Thus, a particle whose sedimentation coefficient is measured at 10-12 sec, i.e. 10 × 10-13 sec, is said to have a sedimentation coefficient of 10 S.

The mathematical bases of sedimentation theory (both rate-zonal and isopycnic) have concerned many authors since the methods were first introduced, and a detailed analysis of this aspect of centrifugation is described in Chapter 2. Many biologists consider that the mathematics included in most discussions of sedimentation theory is esoteric in the extreme, and constitutes a considerable disincentive to their attempting to understand the basic parameters governing fractionations in a centrifugal field. To help counteract this feeling, Chapter 2 is so organized that the basic theories are presented in the text of the chapter with the main mathematical equations involved simply being stated, whereas the detailed derivations of the equations are grouped together in the Appendix to Chapter 2. The use of these equations for the interpretation of data from the analytical ultracentrifuge is explained in Chapter 8, which also gives some indication of the accuracy and reliability of the theory in practical situations. In fact, a considerable amount of valuable quantitative information can be obtained by applying some of these equations to data from experiments done in preparative ultracentrifuges, and the theme is extended in Chapters 5 and 6, which show how these quantitative data can easily be obtained by the use of no more than a little simple arithmetic.

CENTRIFUGATION METHODS

There are three main types of centrifugal fractionation, namely, () differential pelleting (differential centrifugation); () rate-zonal density-gradient sedimentation; and () isopycnic density-gradient sedimentation.

Of these techniques, differential pelleting is the method most commonly used for fractionating material according to size. In this method, the material to be fractionated is initially distributed uniformly throughout the sample solution, which is the sole occupant of the centrifuge tube (). After centrifugation the pellet is enriched in the larger particles of the mixture (). However, the pellet obtained always consists of a mixture of the different species of particle, and it is only the most slowly sedimenting component of the mixture that remains in the supernatant liquid which can be purified by a single centrifugation. The amount of contamination in the pellet can be reduced by washing it (that is, by resuspending and recentrifuging), but this inevitably reduces the yields obtained. Some improvement can be made by, for example, sedimenting the particles through a pad of dense sucrose, although such methods are not universally applicable. An example of a general scheme which is the basis of many used to fractionate cells into their components is outlined in . However, it is not possible to discuss such general schemes in any detail. The actual method which should be used to isolate any particular species of particle depends on a host of independently variable factors, including the tissue from which the particles are to be isolated, the purpose for which they are required, the precise composition of the medium in which the cells are homogenized, and the interaction of other components of the homogenate with the particles of interest. No general scheme can make allowances for the variation in all of these factors, and it is, therefore, much more satisfactory to look at the problems involved from the point of view of each individual species of particle. This is beyond the scope of a book of this kind, and reference should be made to publications such as (2nd edn) and , published by Butterworths in 1972 and 1976, respectively, which deal with the problems involved in isolating the various components of eukaryotic cells from a variety of tissues. The simplicity of differential pelleting methods makes them very attractive for a large number of fractionations on a preparative scale. Moreover, the methods have the advantage that they can be performed with fixed-angle rotors which generally have a higher capacity than the swing-out...