Contents of other Volumes ...................................... xi
Contributors ................................................ xxiii
Preface ....................................................... xiv
PART A. ORGANELLES AND CELLULAR STRUCTURES
Section 1. Isolation: Plasma Membrane, Organelles, and
Cellular Structures
1. Detergent-Resistant Membranes and the Use of
Cholesterol Depletion ........................................ 5
SEBASTIAN SCHUCK, MASANORI HONSHO, AND KAI SIMONS
2. Isolation and Subfractionation of Plasma Membranes to
Purify Caveolae Separately from Lipid Rafts ................. 11
PHILIP O.H, LUCY A. CARVER, AND JAN E. SCHNITZER
3. Immunoisolation of Organelles Using Magnetic Solid
Supports .................................................... 27
RALUCA FLUKIGER-GAGESCU AND JEAN GRUENBERG
4. Purification of Rat Liver Golgi Stacks ...................... 33
YANZHUANG WANG, TOMOHIKO TAGUCHI, AND GRAHAM WARREN
5. Isolation of Rough and Smooth Membrane Domains of
the Endoplasmic Reticulum from Rat Liver .................... 41
JACQUES PAIEMENT, ROBIN YOUNG, LINE ROY, AND JOHN
J.M. BERGERON
6. Purification of COPI Vesicles ............................... 45
FREDRIK KARTBERG, JOHAN HIDING, AND TOMMY NILSSON
7. Purification of Clathrin-Coated Vesicles from Bovine
Brain, Liver, and Adrenal Gland ............................. 51
ROBERT LINDNER
8. Isolation of Latex Bead- and Mycobacterial-Containing
Phagosomes .................................................. 57
MARK KUHNEL, ELSA ANES, AND GARETH GRIFFITHS
9. Isolation of Peroxisomes .................................... 63
ALFRED VOLKL AND H. DARIUSH FAHIMI
10.Isolation of Mitochondria from Mammalian Tissues and
Culturerd Cells ............................................. 69
ERIKA FERNANDEZ-VIZARRA, PATRICIO FERNANDEZ-SILVA,
AND JOSE A. ENRIQUEZ
11.Subcellular Fractionation Procedures and Metabolic
Labeling Using [35SlSulfate to Isolate Dense Core
Secretory Granules from Neuroendocrine Cell Lines ........... 79
SHARON A. TOOZE
12.Preparation of Synaptic Vesicles from Mammalian Brain ....... 85
JOHANNES W. HELL AND REINHARD JAHN
13.Preparation of Proteasomes .................................. 91
KEIJI TANAKA, HIDEKI YASHIRODAS, AND NOBUYUKI
TANAHASHI
14.Preparation of Cilia from Human Airway Epithelial Cells ..... 99
LAWRENCE E. OSTROWSKI
15.Isolation of Nucleoli ...................................... 103
YUN WAH LAM AND ANGUS I. LAMOND
16.Purification of Intermediate-Containing Spliceosomes ....... 109
MELISSA S. JURICA
17.Isolation of Cajal Bodies .................................. 115
YUN WAH LAM AND ANGUS I. LAMOND
18.Replication Clusters: Labeling Strategies for the
Analysis of Chromosome Architecture and Chromatin
Dynamics ................................................... 121
DEAN A. JACKSON, CHI TANG, AND CHRIS DINANT
19.Isolation of Chromosomes for Flow Analysis and Sorting ..... 133
NIGEL P. CARTER
Section 2. Vital Staining of Cells/Organelles
20.Vital Staining of Cells with Fluorescent Lipids ............ 139
TOSHIHIDE KOBAYASHI, ASAMI MAKINO, AND KUMIKO ISHII
21.Labeling of Endocytic Vesicles Using Fluorescent Probes
for Fluid-Phase Endocytosis ................................ 147
NOBUKAZU ARAKI
Section 3. Protein Purification
22.Preparation of Tubulin from Porcine Brain .................. 155
ANTHONY J. ASHFORD AND ANTHONY A. HYMAN
23.Purification of Smooth Muscle Actin ........................ 161
GERALD BURGSTALLER AND MARIO GIMONA
24.Purification of Nonmuscle Actin ............................ 165
HERWIG SCHULER, ROGER KARLSSON, AND UNO LINDBERG
25.Purification of Skeletal Muscle Actin ...................... 173
SEBASTIAN WIESNER
PART B. ASSAYS
Section 4. Endocytic and Exocytic Pathways
26.Permeabilized Epithelial Cells to Study Exocytic
Membrane Transport ......................................... 181
FRANK LAFONT, ELINA IKONEN, AND KAI SIMONS
27.Studying Exit and Surface Delivery of Post-Golgi
Transport Intermediates Using in vitro and Live-Cell
Microscopy-Based Approaches ................................ 189
GERI E. KREITZER, ANNE MUESCH, CHARLES YEAMAN, AND
ENRIQUE RODRIGUEZ-BOULAN
28.Assays Measuring Membrane Transport in the Endocytic
Pathway .................................................... 201
LINDA J. ROBINSON AND JEAN GRUENBERG
29.Microsome-Based Assay for Analysis of Endoplasmic
Reticulum to Golgi Transport in Mammalian Cells ............ 209
HELEN PLUTNER, CEMAL GURKAN, XIAODONG WANG, PAUL
LAPOINTE, AND WILLIAM E. BALCH
30.Cotranslational Translocation of Proteins into
Microsomes Derived from the Rough Endoplasmic
Reticulum of Mammalian Cells ............................... 215
BRUNO MARTOGLIO AND BERNHARD DOBBERSTEIN
31.Use of Permeabilsed Mast Cells to Analyze Regulated
Exocytosis ................................................. 223
GERALD HAMMOND AND ANNA KOFFER
Section 5. Membranes
32.Syringe Loading: A Method for Assessing Plasma Membrane
Function as a Reflection of Mechanically Induced Cell
Loading .................................................... 233
MARK S.F. CLARKE, JEFF A. JONES, AND DANIEL L. FEEBACK
33.Cell Surface Biotinylation and Other Techniques for
Determination of Surface Polarity of Epithelial
Monolayers ................................................. 241
AMI DEORA, SAMIT CHATTERJEE, ALAN D. MARMORSTEIN,
CHIARA ZURZOLO, ANDRE LE BIVIC, AND ENRIQUE
RODRIGUEZ-BOULAN
Section 6. Mitochondria
34.Protein Translocation into Mitochondria .................... 253
SABINE ROSPERT AND HENDRIK OTTO
35.Polarographic Assays of Mitochondrial Functions ............ 259
Y.E. XIONG, PATTI L. PETERSON, AND CHUAN-PU LEE
Section 7. Nuclear Transport
36.Analysis of Nuclear Protein Import and Export in
Digitonin-Permeabilized Cells .............................. 267
RALPH H. KEHLENBACH AND BRYCE M. PASCHAL
37.Heterokaryons: An Assay for Nucleocytoplasmic Shuttling .... 277
MARGARIDAGAMA-CARVALHO AND MARIA CARMO-FONSECA
Section 8. Chromatin Assembly
38.DNA Replication-Dependent Chromatin Assembly System ........ 287
JESSICA K. TYLER
Section 9. Signal Transduction Assays
39.Cygnets: Intracellular Guanosine 3',5'-Cyclic
Monophosphate Sensing in Primary Cells Using
Fluorescence Energy Transfer ............................... 299
CAROLYN L. SAWYER, AKIRA HONDA, AND WOLFGANG
R.G. DOSTMANN
40.Ca2+ as a Second Messenger: New Reporters for Calcium
(Cameleons and Camgaroos) .................................. 307
KLAUS P. HOEFLICH, KEVIN TRUONG, AND MITSUHIKO IKURA
41.Ratiometric Pericam ........................................ 317
ATSUSHI MIYAWAKI
42.Fluorescent Indicators for Imaging Protein
Phosphorylation in Single Living Cells ..................... 325
MORITOSHI SATO AND YOSHIO UMEZAWA
43.In Situ Electroporation of Radioactive Nucleotides:
Assessment of Ras Activity or 32P Labeling of Cellular
Proteins ................................................... 329
LEDA RAPTIS, ADINA VULTUR, EVI TOMAI, HEATHER
L. BROWNELL, AND KEVIN L. FIRTH
44.Dissecting Pathways; in Situ Electroporation for
the Study of Signal Transduction and Gap Junctional
Communication .............................................. 341
LEDA RAPTIS, ADINA VULTUR, HEATHER L. BROWNELL,
AND KEVIN L. FIRTH
45.Detection of Protein-Protein Interactions in vivo
Using Cyan and Yellow Fluorescent Proteins ................. 355
FRANCIS KA-MING CHAN
46.Tracking Individual Chromosomes with Integrated Arrays
of lacop Sites and GVF-lac Repressor: Analyzing
Position and Dynamics of Chromosomal Loci in
Saccharomyces cerevisiae ................................... 359
FRANK R. NEUMANN, FLORENCE HEDIGER, ANGELA TADDEI,
AND SUSAN M. GASSER
Section 10.Assays and Models of in vitro and in vitro
Motility
47.Microtubule Motility Assays ................................ 371
N.J. CARTER AND ROBERT A. CROSS
48.In vitro Assays for Mitotic Spindle Assembly and
Function ................................................... 379
CELIA ANTONIO, REBECCA HEALD, AND ISABELLE VERNOS
49.In vitro Motility Assays with Actin ........................ 387
JAMES R. SELLERS
50.Use of Brain Cytosolic Extracts for Studying Actin-
Based Motility of Listeria monocytogenes ................... 393
ANTONIO S. SECHI
51.Pedestal Formation by Pathogenic Escherichia coli:
A Model System for Studying Signal Transduction to the
Actin Cytoskeleton ......................................... 399
SILVIA LOMMEL, STEFANIE BENESCH, MANFRED ROHDE, AND
JURGEN WEHLAND
52.Listeria monocytogenes: Techniques to Analyze Bacterial
Infection in vitro ......................................... 407
JAVIER PIZARRO-CERDA AND PASCALE COSSART
Section 11.Mechanical Stress in Single Cells
53.Measurement of Cellular Contractile Forces Using
Patterned Elastomer ........................................ 419
NATHALIE Q. BALABAN, ULRICH S. SCHWARZ, AND BENJAMIN
GEIGER
PART C. APPENDIX
54.Resources .................................................. 427
JOSE M. A. MOREIRA AND EMMANUEL VIGNAL
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