protein - dna interactions

Preface
DNA binding proteins are involved in a number of basic cellular pro-
cesses including transcription, DNA replication, transposition, restric-
tion, recombination, and DNA repair. The chapters in this volume cover a
number of important methods, some old and some new, that are now
widely used in the study of DNA binding proteins. These include purifica-
tion and protein characterization, assays of protein-DNA binding and
protein-induced DNA bending which can be used in vitro and in vivo, and
biochemical and genetic methods for probing the structure, energy, and
specificity of protein-DNA interactions. Previous volumes of this series
that contain related methods include 65, 100, 130, 154, 155, and 170.
ROBERT T.
SAUER
xiii
Contributors to Volume 208
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
CHRISTOPHER R. AIKEN (21),
Infectious
Diseases Laboratory, The Salk Institute,
San Diego, California 92186
BRUCE
M. ALBERTS (3),
Department of
Biochemistry and Biophysics, University
of California, San Francisco, San Fran-
cisco, California 94143
JACK BARRY (3),
Department of Biochemis-
try and Biophysics, University of Cali-
fornia, San Francisco, San Francisco,

California 94143
JEREMY
M. BERG (4),
Department of Bio-
physics and Biophysical Chemistry, The
Johns Hopkins University, Baltimore,
Maryland 21205
JAMES U. BOWIE
(27, 29),
Department of
Chemistry and Biochemistry, University
of California, Los Angeles, Los Angeles,
California 90024
RICHARD M. BREYER (27),
Laboratoire
d'lmmuno-Pharmacologie Moleculaire,
Institut Cochin de Genetique Molecu-
laire, Paris, France
THOMAS W. BRUC1E (20),
Department of
Biological Chemistry and Department of
Chemistry and Biochemistry and Molec-
ular Biology Institute, University of Cali-
fornia, Los Angeles, Los Angeles, Cali-
fornia 90024
HENRI BUC (14),
Unitg de Physicochimie
des Macromoldcules Biologiques, Institut
Pasteur, 75724 Paris Cedex 15, France
MALCOLM BUCKLE (14),

Unitd de Physico-
chimie des Macromoldcules Biologiques,
lnstitut Pasteur, 75724 Paris Cedex 15,
France
WLODZIMIERZ BUJALOWSKI (15),
Depart-
ment of Human Biological Chemistry and
Genetics, University of Texas Medical
Branch, Galveston, Texas 77550
RICHARD R. BURGESS (1),
McArdle Labora-
tory for Cancer Research, University of
Wisconsin-Madison, Madison, Wisconsin
53706
JANNETTE CAREY (8),
Department of
Chemistry, Princeton University, Prince-
ton, New Jersey 08544
ARTEMIS E. CHAKERIAN (23),
Department
of Biochemistry and Cell Biology, Rice
University, Houston, Texas 77251
CH1NG-HONG B. CHEN (20),
Department of
Biological Chemistry and Department tff"
Chemistry and Biochemistry and Molec-
ular Biology Institute, University of Cali-
fornia, Los Angeles, Los Angeles, Cali-
fornia 90024
DONALD M. CROTHERS (9),

Department ¢ff~
Chemistry, Yale University, New Haven.
Connecticut 06511
PETER B. DERVAN (24),
Arnold and Mable
Beckman Laboratories of Chemical Syn-
thesis, California Institute of Technology,
Pasadena, California 91125
WENDY J. DIXON (19),
Department of Biol-
ogy, The Johns Hopkins University, Balti-
more, Maryland 21218
MARK DODSON (11),
Department of BiD-
chemistry, Stanford University School cff
Medicine, Stanford, California 94305
BETH A. DOMaROSKI (19),
Center for Medi-
cal Genetics, The Johns Hopkins Medical
Institutions, Baltimore, Maryland 21205
RICHARD n. EBRIGHT (30),
Department
of Chemistry and Waksman Institute,
Rutgers University, New Brunswick, New
Jersey 08855
HARRISON ECHOLS (11),
Department of
Molecular and Cell Biology, University of
California, Berkeley, Berkeley, California
94720

ix
X CONTRIBUTORS TO VOLUME
208
ELISABETH M. EVERTSZ (13), Institute of
Molecular Biology, and Departments of
Chemistry and Biology, University of
Oregon, Eugene, Oregon 97403
MATTHEW A. FISHER (16), Department of
Chemistry, Randolph-Macon College,
Ashland, Virginia 23005
TIM FORMOSA (3), Department of Biochem-
istry, University of Utah, Salt Lake City,
U~ah 84132
ALEXANDRE FRITSCH (14), Unit( de Phy-
sicochimie des Macromol~cules Biologi-
ques, lnstitut Pasteur, 75724 Paris Cedex
15, France
JOSEPH A. GARDNER (23), Department of
Biochemistry and Cell Biology, Rice Uni-
versity, Houston, Texas 77251
MARC R. GARTENBERG (9), Department of
Molecular Biology and Biophysics, Har-
vard University, Cambridge, Massachu-
setts 02138
JOHANNES GEISELMANN (14), Unit~ de Phy-
sicochimie des Macromol~cules Biologi-
ques, Institut Pasteur, 75724 Paris Cedex
15, France
AMY L. GIaSON (31), Department of Biolog-
ical Chemistry, The University of Michi-

gan, Ann Arbor, Michigan 48109
STEPHEN P. GOFF (28), Department of Bio-
chemistry and Molecular Biophysics, Co-
lumbia University College of Physicians
and Surgeons, New York, New York
10032
JAY D. GRALLA (10), Department of Chem-
istry and Biochemistry, and the Molecular
Biology Institute, University of Califor-
nia, Los Angeles, Los Angeles, California
90024
JACK GREENBLATT (3), Banting and Best
Department of Medical Research, Uni-
versity of Toronto, Toronto, Ontario,
Canada
RICHARD I. GUMPORT (21), Department of
Biochemistry, University of Illinois, Col-
lege of Medicine, Urbana, Illinois 61801
JEUNG-HoI HA (16), Department of Bio-
chemistry, University of Wisconsin
Madison, Madison, Wisconsin 53706
JEFFREY J. HAYES (19), Laboratory of
Molecular Biology, National Institutes of
Health, Bethesda, Maryland 20892
WOLFGANG HILLEN (5, 18), lnstitut ffir
Mikrobiolgie und Biochemie, Friedrich-
Alexander Universitiit Erlangen-Nfirn-
berg, D-8520 Erlangen, Germany
ANN HOCHSCHILD (17), Department of Mi-
crobiology and Molecular Genetics, Har-

vard Medical School, Boston, Massachu-
setts 02115
JOEL W. HOCKENSMITH (13), Department
of Biochemistry, University of Virginia
School of Medicine, Charlottesville, Vir-
ginia 22908
JAMES C. Hu (27), Department of Biology,
Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
ANDRZEJ JOACHIMIAK (7), Department of
Molecular Biophysics and Biochemistry,
Yale University, New Haven, Connecti-
cut 06511
JAMES T. KADONAGA (2), Department of Bi-
ology and Center for Molecular Genet-
ics, University of California, San Diego,
La Jolla, California 92093
RACHEL E. KLEVIT (6), Department of Bio-
chemistry, University of Washington,
Seattle, Washington 98195
KENDALL L. KNIGHT (27), Department of
Biochemistry and Molecular Biology,
University of Massachusetts Medical
Center, Worcester, Massachusetts 01655
WILLIAM H. KONIGSBERG (25), Department
of Molecular Biophysics and Biochemis-
try, Yale University School of Medicine,
New Haven, Connecticut 06510
WILLIAM L. KUBASEK (13), Department of
Molecular Biology, Massachusetts Gen-

eral Hospital, Boston, Massachusetts
02114
MICHIO D. KUWABARA (20), Department of
Biological Chemistry and Department of
Chemistry and Biochemistry and Molec-
ular Biology Institute, University of Cali-
fornia, Los Angeles, Los Angeles, Cali-
fornia 90024
CONTRIBUTORS TO VOLUME 208
xi
ARTHUR D. LANDER (12), Department of
Biology and Department of Brain and
Cognitive Sciences, Massachusetts In-
stitute of Technology, Cambridge,
Massachusetts 02139
JUDITH R. LEVIN (19), Department of
Chemistry, The Johns Hopkins Univer-
sity, Baltimore, Maryland 21218
WENDELL A. LIM (12, 27), Department of
Biology, Massachusetts Institute of Tech-
nology, Cambridge, Massachusetts 02139
TIMOTHY M, LOHMAN (15), Department of
Biochemistry and Molecular Biophysics,
Washington University School of Medi-
cine, St. Louis, Missouri 63110
KATHLEEN S. MATTHEWS (23), Department
of Biochemistry and Cell Biology, Rice
University, Houston, Texas 77251
DENISE L. MERKLE (4), Department of
Chemistry, University of Florida, Gaines-

ville, Florida 32611
JEFFREY H. MILLER (26), Department of
Microbiology and Molecular Genetics,
and the Molecular Biology Institute, Uni-
versity of California, Los Angeles, Los
Angeles, California 90024
MICHAEL C. MOSSING (27, 29), Department
of Biological Sciences, University of
Notre Dame, South Bend, Indiana 46556
DALE L. OXENDER (31), Department of
Biotechnology, Park-Davis Research Di-
vision of Warner-Lambert, Ann Arbor,
Michigan 48106
GRACE PARRAGA (6), Department of Bio-
chemistry, Biocenter of the University of
Basel, Basel CH-4056, Switzerland
DAWN A. PARSELL (27), Howard Hughes
Medical Institute, University of Chicago,
Chicago, Illinois 60637
VINAYAKA R, PRASAD (28), Department of
Microbiology and Immunology, Albert
Einstein College of Medicine, Bronx, New
York 10461
M. THOMAS RECORD, JR. (16), Department
of Chemistry and Biochemistry, Univer-
sity of Wisconsin Madison, Madison,
Wisconsin 53706
JOHN F. REIDHAAR-OLsoN (27), Depart-
ment of Biochemistry and Biophysics,
University of California, San Francisco,

San Francisco, California 94143
PASCAL ROUX (14), Unitd de Physicochimie
des Macromoldcules Biologiques, lnstitut
Pasteur, 75724 Paris Cedex 15, France
SELINA SASSE-DWIGHT (10), Department of
Genetics, Star, ford University School
of Medicine, Stanford, California 94305
ROaERT T. SAUER (12, 27, 29), Department
of Biology, Massachusetts Institute ~f
Technology, Cambridge, Massachusetts
02139
KEVIN R. SHOEMAKER (27), Department of
Biology, Massachusetts Institute of Tech-
nology, Cambridge, Massachusetts 02139
THOMAS E. SHRADER (9), Department of Bi-
ology, Massachusetts Institute of Tech-
nology, CamOridge, Massachusetts 02139
PAUL B. SIGLER (7), Department of Molecu-
lar Biophysics and Biochemistry, Howard
Hughes Medical Institute, Yale Univer-
sity, New Haven, Connecticut 06511
DAVID S. SIGMAN (20), Department of Bio-
logical Chemistry, Molecular Biology
Institute, University of California, Los
Angeles, Los Angeles, California 90024
GARY D. STORMO (22), Department of Mo-
lecular, Cellular and Developmental Biol-
ogy, University of Colorado, Boulder,
Colorado 80309
KARLHEINZ TOVAR (5), lnstitut fiir Mikro-

biologie und Biochemie, Friedrich-Alex-
ander Universitiit Erlangen-Niirnberg, D-
6382 Erlangen, Germany
THOMAS D. TULLIUS (19), Department of
Chemistry, The Johns Hopkins Univer-
sity, Baltimore, Maryland 21218
PETER H. YON HIPPEL (13), Institute of
Molecular Biology, and Departments of
Chemistry and Biology, University of
Oregon, Eugene, Oregon 97403
WILLIAM R. VORACHEK (13), Department
of Biochemistry, University of Virginia
School of Medicine, Charlottesville, Vir-
ginia 22908
xii CONTRIBUTORS TO VOLUME 208
MARGARET F. WEIDNER (19), Seattle,
Washington 98109
KENNETH R. WILLIAMS
(25),
Howard
Hughes Medical Institute, Department of
Molecular Biophysics and Biochemistry,
Yale University School of Medicine, New
Haven, Connecticut 06510
ANDREAS WISSMANN (18), BASF Biore-
search Corp., Cambridge, Massachusetts
02139
METHODS IN ENZYMOLOGY
VOLUME I. Preparation and Assay of Enzymes
Edited by

SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME II. Preparation and Assay of Enzymes
Edited by
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME III. Preparation and Assay of Substrates
Edited by
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME IV. Special Techniques for the Enzymologist
Edited by
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME V. Preparation and Assay of Enzymes
Edited by
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME VI. Preparation and Assay of Enzymes
(Continued)
Preparation and Assay of Substrates
Special Techniques
Edited by
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME VII. Cumulative Subject Index
Edited by
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME VIII. Complex Carbohydrates
Edited by
ELIZABETH F. NEUFELD AND VICTOR GINSBURG
VOLUME IX. Carbohydrate Metabolism
Edited by
WILLIS A. WOOD
VOLUME X. Oxidation and Phosphorylation
Edited by

RONALD W. ESTABROOK AND MAYNARD E. PULLMAN
VOLUME XI. Enzyme Structure
Edited by
C. H. W. HIRS
VOLUME XII. Nucleic Acids (Parts A and B)
Edited by
LAWRENCE GROSSMAN AND KIVIE MOLDAVE
xv
xvi METHODS IN ENZYMOLOGY
VOLUME XlII. Citric Acid Cycle
Edited by
J. M. LOWENSTEIN
VOLUME XIV. Lipids
Edited by
J. M. LOWENSTEIN
VOLUME XV. Steroids and Terpenoids
Edited by
RAYMOND B. CLAYTON
VOLUME XVI. Fast Reactions
Edited by
KENNETH KUSTIN
VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)
Edited by
HERBERT TABOR AND CELIA WHITE TABOR
VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)
Edited by
DONALD B. MCCORMICK AND LEMUEL D. WRIGHT
VOLUME XIX. Proteolytic Enzymes
Edited by
GERTRUDE E. PERLMANN AND LASZLO LORAND

VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)
Edited by
KIVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME XXI. Nucleic Acids (Part D)
Edited by
LAWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME XXlI. Enzyme Purification and Related Techniques
Edited by
WILLIAM B. JAKOBY
VOLUME XXIII. Photosynthesis (Part A)
Edited by
ANTHONY SAN PIETRO
VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)
Edited by
ANTHONY SAN PIETRO
VOLUME XXV. Enzyme Structure (Part B)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME XXVI. Enzyme Structure (Part C)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
METHODS IN ENZYMOLOGY xvii
VOLUME XXVII. Enzyme Structure (Part D)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME XXVIII. Complex Carbohydrates (Part B)
Edited by VICTOR GINSBURG
VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)
Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)
Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME XXXI. Biomembranes (Part A)
Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME XXXII. Biomembranes (Part B)
Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX
Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS
VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)
Edited by W1LLIAM B. JAKOBY AND MEIR WILCHEK
VOLUME XXXV. Lipids (Part B)
Edited by JOHN M. LOWENSTEIN
VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)
Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN
VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)
Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN
VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)
Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY
VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and
Organ Systems)
Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY
VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)
Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN
xviii
METHODS IN ENZYMOLOGY
VOLUME XLI. Carbohydrate Metabolism (Part
B)
Edited by
W. A. WOOD
VOLUME XLII. Carbohydrate Metabolism (Part
C)
Edited by

W. A. ;WOOD
VOLUME XLIII. Antibiotics
Edited by
JOHN H. HASH
VOLUME XLIV. Immobilized Enzymes
Edited by
KLAUS MOSBACH
VOLUME XLV. Proteolytic Enzymes (Part B)
Edited by
LASZLO LORAND
VOLUME XLVI. Affinity Labeling
Edited by
WILLIAM B. JAKOBY AND MEIR WILCHEK
VOLUME XLVII. Enzyme Structure (Part E)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME XLVIII. Enzyme Structure (Part F)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME XLIX. Enzyme Structure (Part G)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME L. Complex Carbohydrates (Part C)
Edited by
VICTOR GINSBURG
VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism
Edited by
PATRICIA A. HOFFEE AND MARY ELLEN JONES
VOLUME LII. Biomembranes (Part C: Biological Oxidations)
Edited by

SIDNEY FLEISCHER AND LESTER PACKER
VOLUME LIII. Biomembranes (Part D: Biological Oxidations)
Edited by
SIDNEY FLEISCHER AND LESTER PACKER
VOLUME LIV. Biomembranes (Part E: Biological Oxidations)
Edited by
SIDNEY FLEISCHER AND LESTER PACKER
METHODS IN ENZYMOLOGY xix
VOLUME LV. Biomembranes (Part F: Bioenergetics)
Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME LVI. Biomembranes (Part G: Bioenergetics)
Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME LVII. Bioluminescence and Chemiluminescence
Edited by MARLENE A. DELUCA
VOLUME LVIII. Cell Culture
Edited by WILLIAM B. JAKOBY AND IRA PASTAN
VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)
Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)
Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME 61. Enzyme Structure (Part H)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME 62. Vitamins and Coenzymes (Part
D)
Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT
VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and
Inhibitor Methods)
Edited by DANIEL L. PURICH
VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes
and Complex Enzyme Systems)

Edited by DANIEL L. PURICH
VOLUME 65. Nucleic Acids (Part I)
Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME 66. Vitamins and Coenzymes (Part E)
Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT
VOLUME 67. Vitamins and Coenzymes (Part F)
Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT
XX METHODS IN ENZYMOLOGY
VOLUME 68. Recombinant DNA
Edited by
RAY Wu
VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)
Edited by
ANTHONY SAN PIETRO
VOLUME 70. Immunochemical Techniques (Part A)
Edited by
HELEN VAN VUNAKIS AND JOHN J. LANGONE
VOLUME 71. Lipids (Part C)
Edited by
JOHN M. LOWENSTEIN
VOLUME 72. Lipids (Part D)
Edited by
JOHN M. LOWENSTEIN
VOLUME 73. Immunochemical Techniques (Part B)
Edited by
JOHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 74. Immunochemical Techniques (Part C)
Edited by
JOHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 75. Cumulative Subject Index Volumes XXXI,

XXXIV-LX
Edited by
EDWARD A. DENNIS AND MARTHA G. DENNIS
XXXII,
VOLUME 76. Hemoglobins
Edited by
ERALDO
ANTONINI,
CHIANCONE
LUIGI ROSSI-BERNARDI, AND EMILIA
VOLUME 77. Detoxication and Drug Metabolism
Edited by
WILL1AM B. JAKOBY
VOLUME 78. Interferons (Part A)
Edited by
SIDNEY PESTKA
VOLUME 79. Interferons (Part B)
Edited by
SIDNEY PESTKA
VOLUME 80. Proteolytic Enzymes (Part
C)
Edited by
LASZLO LORAND
METHODS IN ENZYMOLOGY xxi
VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Mem-
branes, I)
Edited by
LESTER PACKER
VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular
Matrix)

Edited by
LEON W. CUNNINGHAM AND DIXIE W. FREDER1KSEN
VOLUME 83. Complex Carbohydrates (Part D)
Edited by
VICTOR GINSBURG
VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoas-
says)
Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile
Apparatus and the Cytoskeleton)
Edited by
DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM
VOLUME 86. Prostaglandins and Arachidonate Metabolites
Edited by
WILLIAM E. M. LANDS AND WILLIAM L. SMITH
VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates,
Stereochemistry, and Rate Studies)
Edited by
DANIEL L. PURICH
VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Mem-
branes, II)
Edited by
LESTER PACKER
VOLUME 89. Carbohydrate Metabolism (Part
D)
Edited by
WILLIS A. WOOD
VOLUME 90. Carbohydrate Metabolism (Part
E)
Edited by

WILLIS A. WOOD
VOLUME 91. Enzyme Structure (Part I)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibod-
ies and General Immunoassay Methods)
Edited by
JOHN J. LANGONE AND HELEN VAN VUNAKIS
xxii
METHODS IN ENZYMOLOGY
VOLUME 93. Immunochemical Techniques (Part F: Conventional Anti-
bodies, Fc Receptors, and Cytotoxicity)
Edited by
JOHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 94. Polyamines
Edited by
HERBERT TABOR AND CELIA WHITE TABOR
VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80
Edited by
EDWARD A. DENNIS AND MARTHA G. DENNIS
VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly
and Targeting (General Methods; Eukaryotes)]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly
and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing
and Recycling)
Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER

VOLUME 99. Hormone Action (Part F: Protein Kinases)
Edited by
JACKIE D. CORBIN AND JOEL G. HARDMAN
VOLUME 100. Recombinant DNA (Part B)
Edited by
RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE
VOLUME 101. Recombinant DNA (Part C)
Edited by
RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE
VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding
Proteins)
Edited
by
ANTHONY R. MEANS AND BERT W. O'MALLEY
VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides)
Edited by
P. MICHAEL CONN
VOLUME 104. Enzyme Purification and Related Techniques (Part C)
Edited by
WILLIAM B. JAKOBY
METHODS IN ENZYMOLOGY
xxiii
VOLUME 105. Oxygen Radicals in Biological Systems
Edited by
LESTER PACKER
VOLUME 106. Posttranslational Modifications (Part
A)
Edited by
FINN WOLD AND KIVIE MOLDAVE
VOLUME 107. Posttranslational Modifications (Part B)

Edited by
FINN WOLD AND KIVIE MOLDAVE
VOLUME 108. Immunochemical Techniques (Part G: Separation and
Characterization of Lymphoid Cells)
Edited by
GIOVANNI DI SABATO, JOHN J. LANGONE, AND
HELEN VAN VUNAKIS
VOLUME 109. Hormone Action (Part I: Peptide Hormones)
Edited by
LUTZ BIRNBAUMER AND BERT W. O'MALLEY
VOLUME 110. Steroids and Isoprenoids (Part A)
Edited by
JOHN H. LAW AND HANS C. RILLING
VOLUME
111.
Steroids and Isoprenoids (Part
B)
Edited by
JOHN H. LAW AND HANS C. RILLING
VOLUME 112. Drug and Enzyme Targeting (Part A)
Edited by
KENNETH J. WIDDER AND RALPH GREEN
VOLUME
pounds
Edited by
113. Glutamate, Glutamine, Glutathione, and Related Com-
ALTON MEISTER
VOLUME 114. Diffraction Methods for Biological Macromolecules (Part
A)
Edited by HAROLD W. WYCKOFF, C. H. W.

HIRS,
AND SERGE N.
TIMASHEFF
VOLUME 115. Diffraction Methods for Biological Macromolecules (Part
B)
Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N.
TIMASHEFF
xxiv METHODS IN ENZYMOLOGY
VOLUME 116. Immunochemical Techniques (Part H: Effectors and Medi-
ators of Lymphoid Cell Functions)
Edited by GIOVANNI
DI
SABATO, JOHN J. LANGONE, AND HELEN VAN
VUNAKIS
VOLUME 117. Enzyme Structure (Part J)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME 118. Plant Molecular Biology
Edited by
ARTHUR WEISSBACH AND HERBERT WEISSBACH
VOLUME 119. Interferons (Part C)
Edited by
SIDNEY PESTKA
VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101
VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technol-
ogy and Monoclonal Antibodies)
Edited by
JOHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 122. Vitamins and Coenzymes (Part G)
Edited by

FRANK CHYTIL AND DONALD B. MCCORMICK
VOLUME 123. Vitamins and Coenzymes (Part H)
Edited by
FRANK CHYTIL AND DONALD B. MCCORMICK
VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides)
Edited by
P. MICHAEL CONN
VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochon-
dria, and Chloroplasts: General Approaches and Transport Systems)
Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochon-
dria, and Chloroplasts: Protonmotive Force)
Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and
Translocation)
Edited by
LESTER PACKER
METHODS IN ENZYMOLOGY XXV
VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and
Molecular Biology)
Edited by
JERE P. SEGREST AND JOHN J. ALBERS
VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biol-
ogy, and Metabolism)
Edited by
JOHN J. ALBERS AND JERE P. SEGREST
VOLUME 130. Enzyme Structure (Part K)
Edited by

C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME 131. Enzyme Structure (Part L)
Edited by
C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and
Cell-Mediated Cytotoxicity)
Edited by
GIOVANNI DI SABATO AND JOHANNES EVERSE
VOLUME 133. Bioluminescence and Chemiluminescence (Part B)
Edited by
MARLENE DELUCA AND WILLIAM D. MCELROY
VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile
Apparatus and the Cytoskeleton)
Edited by
RICHARD B. VALLEE
VOLUME 135. Immobilized Enzymes and Cells (Part B)
Edited by
KLAUS MOSBACH
VOLUME 136. Immobilized Enzymes and Cells (Part C)
Edited by
KLAUS MOSBACH
VOLUME 137. Immobilized Enzymes and Cells (Part D)
Edited by
KLAUS MOSBACH
VOLUME 138. Complex Carbohydrates (Part E)
Edited by
VICTOR GINSBURG
VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-
Binding Proteins)
Edited by

ANTHONY R. MEANS AND P. MICHAEL CONN
VOLUME 140. Cumulative Subject Index Volumes 102-I 19, 121-134
xxvi METHODS IN ENZYMOLOGY
VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids)
Edited by
P. MICHAEL CONN AND ANTHONY R. MEANS
VOLUME 142. Metabolism of Aromatic Amino Acids and Amines
Edited by
SEYMOUR KAUEMAN
VOLUME 143. Sulfur and Sulfur Amino Acids
Edited by
WILLIAM B. JAKOBY AND OWEN GRIFFITH
VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular
Matrix)
Edited by
LEON W. CUNNINGHAM
VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular
Matrix)
Edited by LEON W. CUNNINGHAM
VOLUME 146. Peptide Growth Factors (Part A)
Edited by
DAVID BARNES AND DAVID A. SIRBASKU
VOLUME 147. Peptide Growth Factors (Part B)
Edited by
DAVID BARNES AND DAVID A. SIRBASKU
VOLUME 148. Plant Cell Membranes
Edited by
LESTER PACKER AND ROLAND DOUCE
VOLUME 149. Drug and Enzyme Targeting (Part B)
Edited by

RALPH GREEN AND KENNETH J. WIDDER
VOLUME 150. Immunochemical Techniques (Part K:
In Vitro
Models of B
and T Cell Functions and Lymphoid Cell Receptors)
Edited by
GIOVANNI DI SABATO
VOLUME 151. Molecular Genetics of Mammalian Cells
Edited by
MICHAEL M. GOTTESMAN
VOLUME 152. Guide to Molecular Cloning Techniques
Edited by
SHELBY L. BERGER AND ALAN R. KIMMEL
VOLUME 153. Recombinant DNA (Part D)
Edited by
RAY Wu AND LAWRENCE GROSSMAN
METHODS IN ENZYMOLOGY xxvii
VOLUME 154. Recombinant DNA (Part E)
Edited by
RAY Wu AND LAWRENCE GROSSMAN
VOLUME 155. Recombinant DNA (Part
F)
Edited by
RAY Wu
VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related
Transport: The Na,K-Pump)
Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related
Transport: Calcium, Proton, and Potassium Pumps)

Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 158. Metalloproteins (Part A)
Edited by
JAMES F. RIORDAN AND BERT L. VALLEE
VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action
Edited by
JACKIE D. CORBIN AND ROGER A. JOHNSON
VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose)
Edited by
WILLIS A. WOOD AND SCOTT T. KELLOGG
VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin)
Edited by
WILLIS A. WOOD AND SCOTT T. KELLOGG
VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and In-
flammation)
Edited by
GIOVANNI DI SABATO
VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and In-
flammation)
Edited by
GIOVANNI DI SABATO
VOLUME 164. Ribosomes
Edited by
HARRY F.
NOLLER,
JR., AND KIVIE MOLDAVE
VOLUME 165. Microbial Toxins: Tools for Enzymology
Edited by
SIDNEY HARSHMAN

VOLUME 166. Branched-Chain Amino Acids
Edited by
ROBERT HARRIS AND JOHN R. SOKATCH
xxviii
METHODS IN ENZYMOLOGY
VOLUME 167. Cyanobacteria
Edited by LESTER PACKER AND ALEXANDER N. GLAZER
VOLUME
168.
Hormone Action (Part K: Neuroendocrine Peptides)
Edited by P. MICHAEL CONN
VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A)
Edited by JACEK HAWIGER
VOLUME 170. Nucleosomes
Edited by PAUL M. WASSARMAN AND ROGER D. KORNBERG
VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model
Membranes)
Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and
Characterization)
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport:
Eukaryotic (Nonepithelial) Cells]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport:
Eukaryotic (Nonepithelial) Cells]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 175. Cumulative Subject Index Volumes 135-139, 141-167
VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques

and Dynamics)
Edited by
NORMAN J. OPPENHEIMER AND THOMAS L. JAMES
VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mech-
anism)
Edited by NORMAN J.
OPPENHEIMER AND THOMAS L. JAMES
VOLUME 178. Antibodies, Antigens, and Molecular Mimicry
Edited by JOHN J. LANGONE
METHODS IN ENZYMOLOGY xxix
VOLUME 179. Complex Carbohydrates (Part F)
Edited by
VICTOR G1NSBURG
VOLUME 180. RNA Processing (Part A: General Methods)
Edited by
JAMES E. DAHLBERG AND JOHN N. ABELSON
VOLUME 181. RNA Processing (Part B: Specific Methods)
Edited by
JAMES E. DAHLBERG AND JOHN N. ABELSON
VOLUME 182. Guide to Protein Purification
Edited by
MURRAY P. DEUTSCHER
VOLUME 183. Molecular Evolution: Computer Analysis of Protein and
Nucleic Acid Sequences
Edited by
RUSSELL F. DOOLITTLE
VOLUME 184. Avidin-Biotin Technology
Edited by
MEIR WILCHEK AND EDWARD A. BAYER
VOLUME 185. Gene Expression Technology

Edited by
DAVID V. GOEDDEL
VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen
Radicals and Antioxidants)
Edited by
LESTER PACKER AND ALEXANDER N. GLAZER
VOLUME 187. Arachidonate Related Lipid Mediators
Edited by
ROBERT C. MURPHY AND FRANK A. FITZPATRICK
VOLUME 188. Hydrocarbons and Methylotrophy
Edited by
MARY E. LIDSTROM
VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects)
Edited by
LESTER PACKER
VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applica-
tions)
Edited by
LESTER PACKER
VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport:
Epithelial Cells)
Edited by
SIDNEY FLEISCHER AND BECCA FLEISCHER
XXX METHODS IN ENZYMOLOGY
VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Trans-
port: Epithelial Cells)
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 193. Mass Spectrometry
Edited by JAMES A. MCCLOSKEY
VOLUME 194. Guide to Yeast Genetics and Molecular Biology

Edited by CHRISTINE GUTHRIE AND GERALD R. FINK
VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN
VOLUME 196. Molecular Motors and the Cytoskeleton
Edited by RICHARD B. VALLEE
VOLUME 197. Phospholipases
Edited by EDWARD A. DENNIS
VOLUME 198. Peptide Growth Factors (Part C)
Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO
VOLUME 199. Cumulative Subject Index Volumes 168-174, 176-194
VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays,
Purification, Antibodies, Functional Analysis, Cloning, and Expression)
Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON
VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phos-
phorylation, Protein Kinase Inhibitors, and Protein Phosphatases)
Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON
VOLUME 202. Molecular Design and Modeling: Concepts and Applica-
tions (Part A: Proteins, Peptides, and Enzymes)
Edited by JOHN J. LANGONE
VOLUME 203. Molecular Design and Modeling: Concepts and Applica-
tions (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides,
and Drugs)
Edited by JOHN J. LANGONE
METHODS IN ENZYMOLOGY xxxi
VOLUME 204. Bacterial Genetic Systems
Edited by
JEFFREY H. MILLER
VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Re-
lated Molecules)
Edited by

JAMES F. RIORDAN AND BERT L. VALLEE
VOLUME 206. Cytochrome P450
Edited by
MICHAEL R. WATERMAN AND ERIC F. JOHNSON
VOLUME 207. Ion Channels (in preparation)
Edited by
BERNARDO RUDY AND LINDA E. IVERSON
VOLUME 208. Protein-DNA Interactions
Edited by
ROBERT T. SAUER
VOLUME 209. Phospholipid Biosynthesis (in preparation)
Edited by
EDWARD A. DENNIS AND DENNIS E. VANCE
[1] USE OF POLYETHYLENEIMINE 3
[1] Use of Polyethyleneimine in Purification
of DNA-Binding Proteins
By
RICHARD R. BURGESS
One of the important early steps in purifying a DNA-binding protein is
to separate the protein of interest from cellular nucleic acids. A large
variety of methods have been used, including precipitation with streptomy-
cin sulfate, 1 polyethylene glycol, 2'3 batch treatment of extracts with
DEAE-cellulose,4 and treatment with DNase 1.5 Perhaps the most effective
method involves precipitation with polyethyleneimine.6-8 This chapter will
focus on the use of polyethyleneimine in early stages of the purification
of DNA-binding proteins. It should be noted, however, that the use of
polyethyleneimine in protein purification is not restricted to DNA-binding
proteins and has been used successfully for proteins from both prokaryotic
and eukaryotic sources. The most comprehensive summary of the applica-
tions of polyethyleneimine is found in a review by Jendrisak. 9

Properties of Polyethyleneimine (PEI)
Polyethyleneimine (PEI) is a product of polymerization of ethyleneim-
ine to yield a basic linear polymer with the structure
H2N ( CH2CH2 N ~- ~ CH2CH2 NH 2
Typically, n equals 700-2000 to give a molecular weight range of
30,000-90,000. Since the PKa value of the imino group is 10-11, PEI is a
positively charged molecule in solutions of neutral pH. It has been pro-
duced for over 30 years in large quantities for uses such as a mordant for
the dye industry by BASF (Charlotte, NC) under the trade name, Poly-
min P.
1 M. J. Chamberlin and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 48, 81 (1962).
2 C. Babinet, Biochem. Biophys. Res. Commun. 26, 639 (1967).
3 B. Alberts and G. Herrick, this series, Vol. 21, [11] (1971).
4 W. F. Mangel, Biochim. Biophys. Acta 163, 172 (1974).
5 R. R. Burgess, J. Biol. Chem. 244, 6160 (1969).
6 W. Zillig, K. Zechel, and H. Halbwachs, Hoppe-Seyler's Z. Physiol. Chem. 351, 221
(1970).
7 R. R. Burgess and J. J. Jendrisak, Biochemistry 14, 4634 (1975).
8 j. j. Jendrisak and R. R. Burgess, Biochemistry 14, 4639 (1975).
9 j. j. Jendrisak, in "Protein Purification: Micro to Macro" (R. R. Burgess, ed.), p. 75, Alan
R. Liss, New York, 1987.
Copyright © 1991 by Academic Press. Inc.
METHODS IN ENZYMOLOGY. VOL. 208 All rights of reproduction in any form reserved.
4
PURIFICATION AND CHARACTERIZATION
[1]
For precipitation of nucleic acids and some proteins, it has the desirable
properties of being inexpensive, working well at slightly alkaline pH's
where proteins are usually stable, and rapidly forming finely divided pre-
cipitates which may be centrifuged and resuspended very easily.

Principles of PEI Precipitation
PEI causes precipitation of nucleic acids and acidic proteins at low
ionic strength by forming charge neutralization complexes and cross-
bridges between the complexes. This leads to flocculation.~°
This precipitation is basically a titration. As PEI is added, it becomes
intimately involved in the networked complexes and is thus part of the
precipitate. PEI precipitation thus differs fundamentally from salt or sol-
vent precipitation. In ammonium sulfate precipitation, for example, the
precipitant sequesters H20, decreases H20 available to solvate the protein,
and thus decreases the solubility of the protein, without itself being sub-
stantially precipitated. To illustrate this difference, assume that a given
extract requires 50% saturated ammonium sulfate or 0.2% (w/v) PEI to
achieve 90% precipitation of a given enzyme. If the extract is diluted 10-
fold, it will take higher ammonium sulfate (about 56% saturation) but lower
PEI concentration (0.02%) to achieve a similar 90% precipitation of the
enzyme.
The fraction of the total protein precipitated by PEI will depend on the
pH (at higher pH, more proteins will carry a negative charge and be
precipitated) and on the ionic strength (increasing ionic strength will
weaken PEI-protein interactions). At low ionic strength (0-0.1 M NaCI),
DNA and all acidic proteins will be precipitated. At higher ionic strength
(0.1-1 M NaC1), only DNA and highly negatively charged proteins will
precipitate. At greater than 1 M NaC1, only nucleic acids will precipitate.
DNA-PEI precipitates dissolve at 1.4 to 1.6 M NaC1. There are thus three
basic strategies for using PEI in the purification of DNA binding proteins.
Strategy A: Precipitate with PEI at high ionic strength to remove
nucleic acid. Almost no proteins will bind to DNA or to PEI in 1 M NaCI.
Therefore one can adjust the salt concentration of an extract to 1 M NaCI
and titrate with PEI to achieve precipitation of nucleic acids, leaving the
proteins in the supernatant. This titration can be conveniently followed

by determining the UV absorption spectrum of the supernatants 9 and
adding PEI until the supernatant shows an A280 value greater than the A260 ,
characteristic of protein solutions. While this strategy can be used, it is
not common. One reason is that while nucleic acid is effectively removed,
~0 D. J. Bell, M. Hoare, and P. Dunhill, in "Advances in Biochemical Engineering/Biotech-
nology" (A. Fiechler, ed.), p. 1. Springer-Verlag, Berlin, 1982.
[1] USE OF POLYETHYLENEIM1NE 5
no purification from other proteins is achieved, unlike strategies B or C
(below). Another problem is that if more PEI is added than is needed to
precipitate the nucleic acids, excess PEI will be present in the supernatant
with the proteins. When the supernatant is then diluted or dialyzed to
lower ionic strength, PEI-acidic protein complexes will precipitate. This
excess PEI can be removed by ammonium sulfate precipitation as de-
scribed below.
Strategy B: Precipitate with PEI at low to medium ionic strength to
remove nucleic acids and some proteins, leaving enzyme of interest in
supernatant.
This is like strategy A but, because it is carried out at lower
ionic strength, a substantial number of proteins will be precipitated. The
enzyme of interest will remain in the supernatant and will not only be
nucleic acid free but also will be free of the precipitated acidic proteins.
This strategy works well for neutral or slightly basic DNA-binding proteins
that do not bind to DNA under the ionic conditions of the PEI precipitation.
Strategy C: Precipitate with PEI at lower ionic strength to precipitate
nucleic acids and some proteins and then elute desired enzyme out of
pellet with higher ionic strength.
This strategy is by far the most commonly
used. Generally one chooses a pH of 7.5-7.9 and an ionic strength of 0.1
to 0.2 M NaCI and then performs a PEI titration test to determine the
minimal amount of PEI solution that causes complete precipitation of the

enzyme of interest. The pellet, which contains nucleic acids and some
proteins, is resuspended in a predetermined low ionic strength buffer to
wash out any trapped proteins but not the desired enzyme. The washed
pellet is then resuspended in a buffer of predetermined higher ionic
strength to elute the enzyme, leaving the nucleic acids in the precipitate.
The precise amounts of PEI added and the ionic strength of the wash and
elution buffers are determined empirically as described below. The eluate
contains the enzyme of interest, some other proteins, and a certain amount
of PEI that was originally coprecipitated with the eluted proteins. The PEI
is generally removed by ammonium sulfate precipitation of the protein,
leaving the PEI in the ammonium sulfate supernatant. The ammonium
sulfate pellet is dissolved in buffer and subsequent purification steps are
performed.
Methods and Procedures
Preparing Extracts
Extracts may be prepared in a variety of ways from bacterial,ll animal
and plant 12 cells, or tissues. Generally, the crude extract is centrifuged at
It M. Cull and C. S. McHenry, this series, Vol. 182, [12] (1990).
12 p. Gegenheimer, this series, Vol. 182, [14] (1990).