Table Of ContentSeparation, Detection, and
Characterization of Biological Macromolecules
Series Editor
ARNOLD REVZIN
Department of Biochemistry
Michigan State University
East Lansing, Michigan
Arnold Revzin (Editor). Footprinting of Nucleic Acid-Protein Complexes, 1993.
Gebhard von Jagow and Hermann Schagger (Editors). A Practical Guide to
Membrane Protein Purification, 1994.
A PRACTICAL
GUIDE TO
MEMBRANE
PROTEIN
PURIFICATION
Edited by
GEBHARD VON JAGOW
and
HERMANN SCHAGGER
Zentrum filr Biologische Chemie
Universitatsklinikum Frankfurt
Frankfurt am Main
Federal Republic of Germany
Academic Press
San Diego New York Boston London Sydney Tokyo Toronto
This book is printed on acid-free paper. ©
Copyright © 1994 by ACADEMIC PRESS, INC.
All Rights Reserved.
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or any information
storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc.
A Division of Harcourt Brace & Company
525 B Street, Suite 1900, San Diego, California 92101-4495
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
A Practical guide to membrane protein purification / edited by Gebhard
von Jagow, Hermann Schagger.
p. cm. — (Separation, detection, and characterization of
biological macromolecules)
Includes bibliographical references and index.
ISBN 0-12-725550-8
1. Membrane proteins-Purification. I. Jagow, Gebhard von.
II. Schagger, Hermann. III. Series.
QP552.M44P73 1994
574.19'245-dc20 94-14643
CIP
PRINTED IN THE UNITED STATES OF AMERICA
94 95 96 97 98 99 EB 9 8 7 6 5 4 3 2 1
Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Martin Bodicker (135), Institut fur Biochemie, Heinrich-Heine-Universitat
Dusseldorf, 40225 Dusseldorf, Federal Republic of Germany
Susan K. Buchanan (125), M. R. C. Laboratory of Molecular Biology,
Cambridge CB2 2QH, United Kingdom
Andrea Duchene (141), Institut fur Mikrobiologie, J. W. Goethe-Universitat,
Frankfurt am Main, Federal Republic of Germany
Viktor Geisler (141), Institut fur Mikrobiologie, J. W. Goethe-Universitat,
Frankfurt am Main, Federal Republic of Germany
Achim Kroger (141), Institut fur Mikrobiologie, J. W. Goethe-Universitat,
Frankfurt am Main, Federal Republic of Germany
xi
Xll Contributors
Thomas A. Link (3), Zentrum fur Biologische Chemie, Universitatsklinikum
Frankfurt, 60590 Frankfurt am Main, Federal Republic of Germany
Hermann Schagger (3, 23, 59, 81, 107), Zentrum fur Biologische Chemie,
Universitatsklinikum Frankfurt, 60590 Frankfurt am Main, Federal Republic
of Germany
Georgios Tsiotis1 (149), Max-Planck-Institut fur Biophysik, D-60528 Frankfurt
am Main, Federal Republic of Germany
John E. Walker (125), M. R. C. Laboratory of Molecular Biology, Cambridge
CB2 2QH, United Kingdom
Hanns Weiss (135), Institut fur Biochemie, Heinrich-Heine-Universitat
Diisseldorf, 40225 Diisseldorf, Federal Republic of Germany
Gebhard von Jagow (3), Zentrum fur Biologische Chemie, Universitats-
klinikum Frankfurt, 60590 Frankfurt am Main, Federal Republic of Germany
1 Present address: Maurice E. Miiller Institut for High-Resolution Electron Microscopy, Biocenter
of the University of Basel, CH-4056 Basel, Switzerland.
Preface to the Series
A Practical Guide to Membrane Protein Purification is the second in a series
of laboratory handbooks that address current topics of wide interest in bio-
chemistry and molecular cell biology, to provide bench scientists with state-
of-the-art, usable, information about important experimental techniques. The
series title was chosen to be inclusive, but it is not our intent to try to cover
every possible subject. Emphasis is placed on techniques that have been, or
are expected to be, of central interest for many years.
The design of the volumes is "user-friendly." The fundamentals of each
method are presented, along with clear protocols that should be of immediate
value in typical laboratory situations. In addition to the principles of the
techniques, each book will contain descriptions of other applications for the
methods, including novel approaches where appropriate. Our goal is to ex-
pand the reader's horizons, thus opening new avenues of research.
xiii
xiv Preface to the Series
There are numerous compilations of experimental techniques already on
the market, some more comprehensive than others. Separation, Detection, and
Characterization of Biological Macromolecules is to be distinguished by the quality
and timeliness of its contents, and by the practical value of each volume to
a broad range of working scientists.
Arnold Revzin
Preface to the Volume
Purification of membrane proteins in general seems to be more problematic
than purification of water-soluble proteins, although the methods used are
principally the same. This book is written especially for researchers who are
somewhat familiar with the separation of water-soluble proteins, but who
may not be aware of the pitfalls they may face with membrane proteins.
Detailed descriptions of most of the methods per se are available in numerous
monographs and in the instructions from suppliers of appropriate media.
Therefore, we present these techniques in a concise format and emphasize
the unique features encountered when membrane proteins are involved.
Moreover, this condensed presentation focuses on the principles of the meth-
ods, which should permit investigators new to the field to adapt the techniques
to their particular needs.
xv
xvi Preface to the Volume
The choice of the specific techniques included in this volume, as well as
the emphasis on applications to mitochondrial membrane protein purification,
reflects the authors' preferences. These techniques have wide applicability.
In each case the "basic" version of the method is presented (i.e., the approach
that requires the lowest level of equipment). For example, the transition from
column chromatography using hydrostatic pressure to fast-protein liquid chro-
matography applications, described in Chapter 6, is readily made when the
idiosyncrasies of membrane proteins and the characteristics of the basic tech-
nique are known.
Many of the methods described in this volume are conducive to large-
scale preparations at low cost. In addition, special attention was paid to new
native and denaturing electrophoretic techniques. These methods demand
minimal previous experience in protein isolation. And although they are small-
scale techniques, the protein yield is sufficient for generating antibodies and
for protein sequencing. In many cases these techniques will be the fastest
way to obtain partial protein sequences (e.g., for molecular biologists).
We thank the authors of individual chapters for contributing their isolation
protocols in a form that emphasizes the practical aspects of the techniques.
In addition, we thank Dr. Arnold Revzin, editor for this series, and Dr. Phyllis
B. Moses, Senior Acquisitions Editor at Academic Press, for their help in
transforming the manuscripts into a usable guide.
Gebhard von Jagow
Hermann Schdgger
Abbreviations
Ant antimycin DTT dithiothreitol
APS ammonium persulfate EDTA ethylenediaminetetraacetic acid
BCA bicinchoninic acid FPLC fast protein liquid
Bis N, N' -methylene bisacrylamide chromatography
BN-PAGE blue native polyacrylamide gel HIC hydrophobic interaction
electrophoresis chromatography
C12-M dodecyl-0-D-maltoside HLB hydrophobic lipophilic balance
(synonymous with DM and HPLC high-performance liquid
LM) chromatography
CHAPS 3-[(3-cholamido)- IEF isoelectric focusing
dimethylammonio]-l- IMAC immobilized metal affinity
propanesulfonic acid chromatography
CM carboxymethyl ISP "Rieske" iron-sulfur protein of
CMC critical micellar concentration complex III
CMT critical micellar temperature LM lauryl-/3-D-maltoside
CNBr cyanogen bromide (synonymous with DM and
CN-PAGE colorless native polyacrylamide C12-M)
gel electrophoresis MES 2-(N-morpholino)ethanesulfonic
Complex I NADH:ubiquinol oxidoreductase acid
Complex II succinateiubiquinol MOPS 3-(N-morpholino)propanesulfonic
oxidoreductase acid
Complex III ubiquinokcytochrome c M relative molecular mass ratio
r
oxidoreductase PAGE polyacrylamide gel
Complex IV cytochrome c oxidase electrophoresis
Complex V F^-ATP synthase PEG polyethylene glycol
Dl complex complex III, missing the 6.4 kDa pi isoelectric point
protein subunit PMSF phenylmethylsulfonyl fluoride
D2 complex complex III, missing the 6.4 kDa PVDF polyvinylidene difluoride
and the ISP subunit RPC reversed-phase chromatography
D2-Ant D2 complex with bound SDS-PAGE sodium dodecyl
antimycin sulfate-polyacrylamide gel
DFP diisopropyl fluorophosphate electrophoresis
DM dodecyl-^-D-maltoside TEMED N,N,N',N' -tetramethyl-
(synonymous with LM and ethylenediamine
C12-M) Tricine [N-tris(hydroxymethyl)]-
DMSO dimethyl sulfoxide methylglycine
DTE dithioerythritol
xvii
