Centrifugal Separations in Molecular and Cell Biology

Contents


1 Ιntroduction: Principles and Practices of Certification

Basic Concepts of Sedimentation Theory

Centrifugation Methods

Designing Centrifugation Experiments

Care of Centrifuges and Rotors


2 The Bases of Centrifugal Separations

Comparison of Zonal Centrifugation With Complementary Techniques

Differential Versus Zonal Centrifugation

Theory of Rate-Zonal Experiments

Sedimentation Coefficient

Location of the Centroid

Resolution in Velocity Experiments

Isopycnic Zonal Experiments

Time to Reach Equilibrium

Resolution of Isopycnic Zones

Interaction Between Solutes in Isopycnic Experiments

Statistical Estimation of Molecular Parameters

S-Values

Statistical Procedures

Simulation of Zonal Experiments

Appendix

Relationships Between s and Radial Co-Ordinates

Convective Instabilities in Zones

Isopycnic Equilibrium

Least-Squares Analysis For Curvilinear Polynomials

References


3 Practical Aspects of Rate-Zonal Centrifugation

Experimental Design

Gradient Materials

Simple Sugars

Glycerol

Polysaccharides

Proteins

Deuterium Oxide

Mixed-Solute Gradients

Resolution and Gradient Shape

Resolution

Design of Gradients

Gradient Generation

Loading, Running and Analyzing Gradients

Sample Loading

Centrifugation Conditions

Unloading and Analysis

Appendix I: Density and Viscosity of Sucrose Solutions

Appendix Ii: Density and Viscosity of Glycerol Solutions

References


4 Fractionations in Zonal Rotors

Design and Operation of Batch-Type Rotors

Dynamic Loading and Unloading

Statically-Loaded (Unloaded) Rotors

Gradients and Sample Preparation

Fractionations in Batch-Type Rotors

Separations of Different Cell Types

Separation of Cells According to Position in The Cell Cycle

Nuclei and Nuclear Membranes

Mitochondria, Lysosomes and Peroxisomes

Plasma Membrane and Endoplasmic Reticulum

Ribosomes and Polysomes

Nucleic Acids

Cell Surface Coat (Glycocalyx)

Plasma Lipoproteins

Viruses

Continuous-Flow Zonal Centrifugation

Design and Operation of Rotors

Fractionation With B-Type Rotors

Fractionation With J- and K-Series Rotors

Centrifugal Elutriation

References 1


5 Rate-Zonal Centrifugation: Quantitative Aspects

Some Important Underlying Assumptions

Guide For The Computerized Calculation of Sedimentation Coefficients

Guide For The Manual Calculation of Sedimentation Coefficients

Accuracy of Sedimentation Coefficients Calculated From Density-Gradient Data Using Large-Scale Zonal Rotors

Some Important Sources of Error

Other Analytical Uses

Appendix I: FORTRAN Program For Calculating Sedimentation Coefficients

Appendix Ii: Calculating Radius-Volume Relationships

Appendix Iii: Sedim Values For Various Particle Densities

Appendix Iv: Calculating The Natural Logarithm of the Rotor Radius

References


6 Isopycnic Centrifugation in Ionic Media

Basic Procedures

Optimum Design of Buoyant Density-Gradient Separations

Equilibrium Gradients

Non-Equilibrium Preformed Gradients

Resolving Power of Gradients

Fractionation of Gradients

Buoyant Density-Gradient Solutes

Data From Isopycnic Gradients

Separations By Isopycnic Banding

Large-Scale Preparative Procedures

Isolation of DNA

Isolation of RNA

Isolation of Protein

References


7 Isopycnic Centrifugation in Non-Ionic Media

General Techniques

Density-Gradient Solutes

Monosaccharides and Disaccharides

Polysaccharides

Lodinated Compounds

Colloidal Silica

Other Gradient Media

References


8 Analytical Ultracentrifugation

History and Scope

Instrumentation

Optical Systems

Analytical Ultracentrifuge Cells

Sedimentation Equilibrium Analysis

Sedimentation Equilibrium in Two-Component Systems

Sedimentation Equilibrium Analysis of Associating Systems

Sedimentation Equilibrium in Multi-Component Systems

Sedimentation Velocity Analysis

Areas of Application

Measurement of Sedimentation Coefficient

Use of Sedimentation Coefficients in Molecular-Weight Determination

Sedimentation Velocity Analysis and Molecular Shape

Large Molecule-Small Molecule Interactions

Large Molecule-Large Molecule Interactions

Conclusions

References


9 Characteristics of Ultracentrifuge Rotors and Tubes

Designs and Types of Rotor

Materials

Types of Rotor

Stability of Rotors

Centrifuge Tubes and Caps

Tubes

Sealing Caps For Tubes

Derating Rotors

Routine Maintenance of Rotors

Appendix: Physical Dimensions and Characteristics of Rotors

The Relationship Between Radius and Volume of Zonal Rotors

Index