Laboratory techniques in biochemistry and molecular biology 5 LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY Volume 5 Edited by T.S. WORK N.I.M.R., Mill Hill, London - E. WORK Imperial College, London - Advisory board G. POPJAK - U.C.L.A. S. BERGSTROM - Stockholm K. BLOCH - Haroard University P. SIEKEVITZ - Rockejeller University E. SMITH - U.C.L.A. E.C. SLATER -Amsterdam NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM OXFORD AMERICAN ELSEVIER PUBLISHING CO., INC. - NEW YORK Part I Brian W. Fox TECHNIQUES OF SAMPLE PREPARATION OF LIQUID SCINTILLATION COUNTING Part I1 P.G. Righetti and J.W. Drysdale ISOELECTRIC FOCUSING 1976 NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM OXFORD AMERICAN ELSEVIER PUBLISHING CO., INC. -NEW YORK 0 1976 North-Holland Publishing Companj) All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means. electronic, mechanical, photocopyiny, recording or otherwise, without the prior permission of the copyright owner. ISBN North-Holland - series: 0 7204 4200 I - volume 5 :0 7204 421 9 2 ISBN American Elseilier: 0 444 11216 2 Published by: NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM ' OXFORD Sole distributors for the U.S.A. and Canada: AMERICAN ELSEVIER PUBLISHING COMPANY, INC. 52 VANDERBILTA VENUE, NEW YORK, N.Y. 10017 Printed in The Netherlands Editor’s preface Progress in research depends upon development of technique. No matter how important the cerebral element may be in the planning of experiments, a tentative hypothesis cannot be converted into an accepted fact unless there is adequate consciousness of the scope and limitation of existing techniques; moreover, the results may be mean- ingless or even positively misleading if the technical ‘know how’ is in- adequate. During the past ten or fifteen years, biochemical methods have become specialized and sophisticated to such a degree that it is now difficult for the beginner, whether undergraduate, graduate or special- ist in another field, to grasp all the minor but important details which divide the successful from the unsuccessful experiment. In order to cope with this problem, we have initiated a new series of Laboratory Mhnuals on technique. Each manual is written by an expert and is designed as a laboratory handbook to be used at the bench. It is hoped that use of these manuals will substantially reduce or perhaps even remove that period of frustration which so often precedes the successful transplant of a specialized technique into a new en- vironment. In furtherance of this aim, we have asked authors to place special emphasis on application rather than on theory; nevertheless, each manual carries sufficient history and theory to give perspective. The publication of library volumes followed by pocket paperbacks is an innovation in scientific publishing which should assist in bringing these manuals into the laboratory as well as into the library. In under- V VI EDITORS’ PREFACE taking the editing of such a diverse series, we have become painfully conscious of our own ignorance but have been encouraged by our board of advisers to whom we owe many valuable suggestions and, of course, by our authors who have co-operated so willingly and have so patiently tolerated our editoral intervention. T.S.& E. Work Editors Contents of parts I and I1 PART I TECHNIQUEOSF SAMPLE PREPARATION FOR LIQUIDS CINTILLATION COUNTING. Brian W . Fox . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . 10 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Chapter 1. General principles of liquid scintillation spectrometry . . . . . . . 13 Chapter 2 . Counting systems . . . . . . . . . . . . . . . . . . . . . . 41 Chapter 3. Preprocessing techniques: general aims and criteria . . . . . . . . 86 Chapter 4 . Animal tissue processing . . . . . . . . . . . . . . . . . . . 107 Chapter 5 . Botanical aspects . . . . . . . . . . . . . . . . . . . . . . . . 142 Chapter 6. Cell cultures . . . . . . . . . . . . . . . . . . . . . . . . 149 Chapter 7. Extracts and chromatographic eluates . . . . . . . . . . . . . 161 Chapter 8. Macromolecules . . . . . . . . . . . . . . . . . . . . . . . 173 Chapter 9 . Electrophoresis, centrifugation and chromatography on solid supports 184 Chapter 10. Inorganic applications . . . . . . . . . . . . . . . . . . . . 199 Chapter 11. Quench correction methods, multiple isotope counting and data evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Chapter 12 . Geophysics and archaeology . . . . . . . . . . . . . . . . . 244 Chapter 13 . Miscellaneous applications and future prospects . . . . . . . . 254 Appendix I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 Appendix I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Appendix I11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 Appendix IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 Appendix V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 VII VIIl CONTENTS OF PARTS I AND 11 PART 11 ISOELECTRICF OCUSINPG..G . Righetti and J.W. Drysdale . . . . . . . . . . 335 Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Chapter 1 . Theory and fundamental aspects of IEF . . . . . . . . . . . . 341 Chapter 2. Preparative IEF . . . . . . . . . . . . . . . . . . . . . . . 377 Chapter 3 . Analytical IEF . . . . . . . . . . . . . . . . . . . . . . . 419 Chapter 4 . General experimental aspects . . . . . . . . . . . . . . . . . 5 01 Chapter 5 . Applications of IEF . . . . . . . . . . . . . . . . . . . . . 527 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573 Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 TECHNIQUES OF SAMPLE PREPARATION FOR LIQUID SCINTILLATION COUNTING Brian W. Fox Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manehester, England