1st Edition

Basic Principles of Analytical Ultracentrifugation




ISBN 9781498751155
Published December 18, 2015 by CRC Press
302 Pages 22 Color & 62 B/W Illustrations

USD $115.00

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Book Description

Analytical ultracentrifugation (AUC) can supply rich information on the mass, shape, size distribution, solvation, and composition of macromolecules and nanoscopic particles. It also provides a detailed view of their reversible single- or multi-component interactions over a wide range of affinities. Yet this powerful technique has been hard to master in mainstream molecular sciences due to a lack of comprehensive books on the subject.

Filling this gap in the literature of biophysical methodology, Basic Principles of Analytical Ultracentrifugation explains the fundamentals in the theory and practice of AUC. The book provides you with up-to-date experimental information to confidently practice AUC. You will understand the basic concepts, full potential, and possible pitfalls of AUC as well as appreciate the current relevance of past work in the field.

The book first introduces the basic principles and technical setup of an AUC experiment and briefly describes the optical systems used for detection. It then explores the ultracentrifugation experiment from a macromolecular standpoint, offering a detailed physical picture of the sedimentation process and relevant macromolecular parameters. The authors present important practical aspects for conducting an experiment, including sample preparation, data acquisition and data structure, and the execution of the centrifugal experiment. They also cover instrument calibration and quality control experiments.

Table of Contents

Analytical Ultracentrifugation Basics
PHYSICAL PRINCIPLES
DETECTION PRINCIPLES

The Sedimenting Particle
PARTICLE-SOLVENT INTERACTIONS
PARTICLE-PARTICLE INTERACTIONS
SEDIMENTATION EFFECTS ON MACROMOLECULAR MIGRATION

Sample Preparation and Ancillary Characterization
COMMON SAMPLE REQUIREMENTS
CHOICE OF SOLVENT AND BUFFER
DETERMINING THE PARTIAL-SPECIFIC VOLUME

Data Acquisition
RADIAL DIMENSION
TEMPORAL DIMENSION
SIGNAL DIMENSION
SPECTRAL DIMENSION

The Sedimentation Experiment
THE IMPORTANCE OF AVOIDING CONVECTION
ROTORS AND SAMPLE CELL ASSEMBLIES
TEMPERATURE
VACUUM
ROTOR SPEEDS
STARTING THE RUN
COMMENCING DATA ACQUISITION AND RUN DURATION
STOPPING THE RUN

Control and Calibration Experiments
DATA DIMENSIONS
STANDARD CONTROL EXPERIMENTS

Appendix A: Macromolecular Partial-Specific Volumes
Appendix B: Solvent Properties
Appendix C: Refractive Index Increments
Appendix D: Solution Column

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Author(s)

Biography

Peter Schuck is an Earl Stadtman Tenure‐Track Investigator and Chief of the Dynamics of Macromolecular Assembly Section in the Laboratory of Cellular Imaging and Macromolecular Biophysics at the National Institute of Biomedical Imaging and Bioengineering, U.S. National Institutes of Health. He obtained his Ph.D. from the Goethe‐University Frankfurt am Main, where he worked on interactions of integral proteins of the erythrocyte membrane using analytical ultracentrifugation.

Huaying Zhao is a Staff Scientist in the Dynamics of Macromolecular Assembly Section in the Laboratory of Cellular Imaging and Macromolecular Biophysics at the National Institute of Biomedical Imaging and Bioengineering, U.S. National Institutes of Health. She received a Ph.D. in chemistry from the University of Mississippi, with a specialization in protein biochemistry. Her main research interests are the development of biophysical methodology for characterizing a variety of macromolecules, including proteins, nucleic acids, polymers, and nanoparticles.

Chad A. Brautigam is an Associate Professor in the Department of Biophysics at The University of Texas Southwestern Medical Center. He earned a Ph.D. in biophysics from Yale University. His research focuses on the structures and functions of lipoproteins of pathogenic bacteria. He is also interested in improving the analysis and presentation of biophysical data.

Rodolfo Ghirlando is a Staff Scientist in the Laboratory of Molecular Biology at the National Institute of Diabetes and Digestive and Kidney Diseases, U.S. National Institutes of Health. He earned his Ph.D. from the Weizmann Institute of Science. His research interests include the study of the in vivo chromatin structure at the 30‐nm fiber level and, more generally, the development of hydrodynamic methodology for the study of challenging biomacromolecular interactions.

Reviews

"This state-of-the-art book is very well written and timely. It will be valuable to both experienced and new practitioners in the use of the analytical ultracentrifuge."
—Geoff Howlett, The University of Melbourne

"This eminently readable book tells the outcome — to present date — of a journey of scientific discovery … Sedimentation analysis of macromolecular systems is the field in which Dr. Peter Schuck and his fellow investigators have been involved throughout the present century and to which they have made contributions not only through their own laboratories’ investigations, but through the widespread use of their algorithms and programs by scientists throughout the world. … this book will be as vital a tool for novices as for senior workers seeking guidance on the more recondite areas of analysis. … If you already know about sedimentation analysis, you will find interest and information alike in its pages. If you are a newcomer to the area, you should take advantage of the opportunity to read a well-written and authoritative account, and become excited by its possibilities."
—From the Foreword by Arthur J. Rowe, Nottingham