Current Protocols in Cell Biology

3178 Pages·2004·41.378 MB·English

Wiley InterScience: Reference Work: Current Protocols in Cell Biology
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Cell biologists use a wide variety of techniques from many disciplines to explore the world of the cell and its
structure and function. Because of this diversity, it is important to have a reputable resource for methods to
use in their investigations. Current Protocols in Cell Biology is part of the collection of Current Protocols Tell us about yourself
for a chance to win an
manuals known for their scope and reliability in providing resources for new as well as tried-and-true iPod!
methods. This book focuses on techniques particularly applicable to cell biology.
Current Protocols in Cell Biology is a collection of protocols written by the scientists who use those How often do you use Wiley InterScience?
methods and designed to meet the needs of both the beginning investigator as well as those who are
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experienced but are expanding the scope of their research to use new methods. The protocols are detailed
guides—complete with materials lists, step-by-step procedures, annotations, illustrations, and commentary Less than once per month
—to the successful completion of the method. Anyone with a basic knowledge of the principles of good
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laboratory technique should be able to follow these protocols regardless of their level of experience. The up to once per month
scope of the manual is wide (and growing) covering cell culture, subcellular fractionation to isolate and
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study specific organelles, various types of microscopy, labeling, cell adhesion, cell and organelle motility,
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Journals
Edited by:
Books
Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada
Reference Works
Series Editor: Kathy Morgan; [email protected] Databases
Current Protocols
Two Volumes
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0-471-24108-3 - Loose-leaf
0-471-24105-9 - CD-ROM
Foreword
Kai Simons1
1 EMBL-Heidelberg, Heidelberg, Germany
Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, Kenneth M. Yamada (eds.)
Current Protocols in Cell Biology
Copyright © 2004 John Wiley & Sons, Inc. All rights reserved.
DOI: 10.1002/0471143030.cbfores00
Online Posting Date: May, 2004
Print Publication Date: October, 1998

In 1896, E.B. Wilson published his classic textbook The Cell in Development and Inheritance, summarizing the 60 years of
biological research since Schleiden and Schwann formulated their cell theory in 1839. He concluded that the key to all ultimate
biological problems must “in the last analysis be sought in the cell.” At the turn of the century biologists had already, by analyzing a
multitude of different organisms, arrived at an understanding that the basic design of cells in all living organisms was similar. This
startling insight was derived from careful observation of cellular behavior and structure using such simple tools as light microscopy
combined with different staining methods. In this century, biochemists and molecular geneticists have probed into the secrets of
cellular metabolism and of gene structure and function, and unraveled many of the underlying principles. While these approaches
have been gloriously successful, one consequence of their powerful reductionism has been that biochemists and molecular biologists,
more or less by necessity, lost track of the cell. Cell biologists, on the other hand, have continued to use microscopic methods,
powerfully supplemented by electron microscopy, and cell biology has become a discipline of cell and tissue morphology. Moreover,
moving against the tide, many cell biologists emphasized diversity even as molecular biologists were unfolding the principles of
biological unity. Thus, until the 1970s cell biology did not occupy center stage in biological research. In the last two decades of this
century, the pendulum has been swinging back, and the cell has once again come to the fore. It is obvious that a more comprehensive
analysis of cellular functions is necessary to understand the basic design of how molecular processes function in the cell. Little is
known about how cells manage to generate and maintain their complex organization in different cell types and tissues. However,
these questions are now attracting increasing attention in biomedical research.
The experimental complexity facing molecular cell biology requires a multitude of different techniques. One unique feature of the
new generation of cell biology methods is that they must enable the investigator to study function in the correct cellular context. This
can be done by reconstituting the cellular context in vitro, as when one couples the translation of secretory proteins with translocation
across the membrane of the endoplasmic reticulum, or by performing detailed investigations in living cells, such as analysis of the
dynamic behavior of proteins biosynthetically labeled with derivatives of green fluorescent protein. Obviously, the new
methodologies are a combination of biochemical, genetic, and morphological techniques. In addition, considerable input derives from
new instrumentation in microscopy and spectroscopy that is revolutionizing cell biological research. Processes that only 10 years ago
seemed inaccessible are now yielding to experimental attack.
It is in this setting that Current Protocols in Cell Biology, available in both print and CD-ROM formats, is most welcome. The editors
of the manual have done an admirable job of compiling the relevant cell biological methods. Moreover, one essential feature of the
Current Protocols series is that the manuals are continually updated and expanded, since the development of effective methodology
requires a great deal of feedback. Regular updates of new and modified techniques are especially important in a dynamic field such as
cell biology in order to keep investigators in touch with continuously changing and improving methods.
Kai Simons
EMBL-Heidelberg

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Copyright© 1999-2005 John Wiley & Sons, Inc. All rights reserved.
Preface
Juan S. Bonifacino1, Mary Dasso1, Joe B. Harford2, Jennifer Lippincott-Schwartz1, Kenneth M. Yamada3
1 National Institute of Child Health and Human Development, Bethesda, Maryland
2 National Cancer Institute, Bethesda, Maryland
3 National Institute of Dental and Craniofacial Research, Bethesda, Maryland
Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, Kenneth M. Yamada (eds.)
Current Protocols in Cell Biology
Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.
DOI: 10.1002/0471143030.cbprefs00
Online Posting Date: May, 2001
Print Publication Date: October, 1998

The universe of cell biology is expanding. One way to sense this expansion is to attend a major meeting dedicated to the field, such as
that of the American Society for Cell Biology. The poster sessions allow one to take a leisurely stroll through the exhibits and, even
without stopping to look at any single poster, get a very good visceral feel for the scope of cell biology. For those who have taken
such a stroll periodically over the past decade or two, the dynamic nature of cell biology and its explosive growth have been
obvious—there is simply more and more about more and more. Once upon a time, poster sessions were dominated by images of cells
fixed (literally) and captured in black and white by electron microscopists. While the electron microscope continues to contribute to
our understanding of cell architecture, more recent poster sessions have witnessed the emergence of row upon row of television
monitors where one can view computer-enhanced video images of living cells captured in the not-so-living colors of rhodamine red,
fluorescein yellow, and the characteristic green that gives the now-famous fluorescent protein its name. Freeze-etching has been
joined by freeze-frame. Furthermore, interspersed among the television sets and whirring VCRs are posters that document detailed
molecular characterizations of a vast array of biochemical components that comprise the diverse cell types being investigated. Other
posters record the latest genes to fall to the seemingly relentless onslaught of the cloners and sequencers, and still others offer
functional insights obtained by knocking out these genes. Every point of the compass has its blot! Genetic studies in yeast, flies, and
worms abound on the one hand, and on the other, a growing number of the presentations form bridges between basic research in cell
biology and the practice of clinical medicine.
Clearly, the scientists who today identify themselves as cell biologists are a diverse community, and great value resides in that
diversity. Borders that once separated research disciplines have faded, and cell biologists have come to appreciate that no single
approach in isolation will open the profound mysteries of the cell. New techniques and new technologies come alongside the tried-
and-true as the tools of cell biology expand along with the field. It is this changing face of cell biology and its methodologies that
represented the greatest challenge in pulling together Current Protocols in Cell Biology. A foundational question addressed by the
editors of this work involved where to draw the boundaries around the field of cell biology. Our decision has been to refuse to draw
such boundaries—they are artificial at best and counterproductive at worst. Instead, we will attempt in this effort to match the
diversity of our field. We will include “classic” methods that remain valuable tools for the modern cell biologist and will also provide
protocols that we believe are destined to become tomorrow's classics.
There is no reason to suspect that the expanding universe of cell biology will cease expanding anytime soon. Indeed, part of the thrill
of being a cell biologist is being constantly surprised by new innovations and discoveries. As a consequence, however, our
community needs a reliable and user-friendly source of laboratory methods that is as expansive as the field itself. To begin to address
this need, we have assembled a series of protocols that cover many aspects of cell biology. While this set of methods is incomplete, it
can be considered a “starter toolbox” that includes many of the most versatile and essential instruments of our trade. This collection
will expand as regular quarterly supplements are added to Current Protocols in Cell Biology. Through these supplements, the editors
will endeavor to make the toolbox increasingly more useful over time. Because of the rapid pace of innovation and discovery in cell
biology, we are expecting that our efforts will be met with some degree of very pleasant frustration as we strive to keep up with this
fast-moving and exciting field.

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Copyright© 1999-2005 John Wiley & Sons, Inc. All rights reserved.
How to Use This Manual
Preface
Juan S. Bonifacino1, Mary Dasso1, Joe B. Harford2, Jennifer Lippincott-Schwartz1, Kenneth M. Yamada3
1 National Institute of Child Health and Human Development, Bethesda, Maryland
2 National Cancer Institute, Bethesda, Maryland
3 National Institute of Dental and Craniofacial Research, Bethesda, Maryland
Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, Kenneth M. Yamada (eds.)
Current Protocols in Cell Biology
Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.
DOI: 10.1002/0471143030.cbprefs00
Online Posting Date: May, 2001
Print Publication Date: October, 1998

How to Use This Manual
Format and Organization
This publication is available in both looseleaf and CD-ROM format. For looseleaf purchasers, a binder is provided to accommodate
the growth of the manual via the quarterly update service. This format allows easy insertion of new pages, units, and chapters that are
added. The index and table of contents are updated with each supplement. CD-ROM purchasers receive a completely new disc every
quarter and should dispose of their outdated discs. The material covered in the two versions is identical.
Subjects in this manual are organized by chapters, and protocols are contained in units. Protocol units, which constitute the bulk of
the book, generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for
unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Other units present more general
information in the form of explanatory text with no protocols. Overview units contain theoretical discussions that lay the foundation
for subsequent protocols. Other discussion units present more general information.
Page numbering in the looseleaf version reflects the modular arrangement by unit; for example, page 1.2.3 refers to Chapter 1 (Cell
Culture), UNIT 1.2 (Media for Culture of Mammalian Cells), page 3 of that particular unit.
Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-
references among units are used and recipes for common reagents are supplied in APPENDIX 2A. Cross-referencing helps to
ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw
materials and analyze results.
Introductory and Explanatory Information
Because this publication is first and foremost a compilation of laboratory techniques in cell biology, we have included explanatory
information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with special overview units
that describe the state of the art of the topic matter and provide a context for the procedures that follow. Chapter and unit
introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe
what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background
information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters,
troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key
references to inform users of particularly useful background reading, original descriptions, or applications of a technique.
Protocols
Many units in the manual contain groups of protocols, each presented with a series of steps. One or more basic protocols are
presented first in each unit and generally cover the recommended or most universally applicable approaches. Alternate protocols are
provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a
variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe
additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because
they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol
steps.
Reagents and Solutions
Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions,
others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions
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How to Use This Manual
are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is
important to note that the names of some of these special solutions might be similar from unit to unit (e.g., RIPA buffer) while the
recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used
stock solutions and buffers are provided once in APPENDIX 2A. These universal recipes are cross-referenced parenthetically in the
materials lists rather than repeated with every usage.
Commercial Suppliers
Throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, and equipment. In some
cases, the noted brand has been found to be of superior quality or it is the only suitable product available in the marketplace. In other
cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an
aid to the novice in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with
substituting their own favorite brands.
Addresses, phone numbers, and facsimile numbers of all suppliers mentioned in this manual are provided in the SUPPLIERS
APPENDIX.
Safety Considerations
Anyone carrying out these protocols may encounter the following hazardous or potentially hazardous materials: (1) radioactive
substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Check
the guidelines of your particular institution with regard to use and disposal of these hazardous materials. Although cautionary
statements are included in the appropriate units, we emphasize that users must proceed with the prudence and precaution associated
with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations.
Animal Handling
Many protocols call for use of live animals (usually rats or mice) for experiments. Prior to conducting any laboratory procedures with
live subjects, the experimental approach must be submitted in writing to the appropriate Institutional Animal Care and Use
Committee (IACUC) or must conform to appropriate governmental regulations regarding the care and use of laboratory animals.
Written approval from the IACUC (or equivalent) committee is absolutely required prior to undertaking any live-animal studies.
Some specific animal care and handling guidelines are provided in the protocols where live subjects are used, but check with your
IACUC or governmental guidelines to obtain more extensive information.
Reader Response
Most of the protocols included in this manual are used routinely in the authors' laboratories. These protocols work for them; to make
them work for you they have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries
to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. Consequently, a
self-mailing reader-response survey can be found at the back of the manual (and is included with each supplement); we encourage
readers to send in their comments.

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Copyright© 1999-2005 John Wiley & Sons, Inc. All rights reserved.
Acknowledgments
Preface
Juan S. Bonifacino1, Mary Dasso1, Joe B. Harford2, Jennifer Lippincott-Schwartz1, Kenneth M. Yamada3
1 National Institute of Child Health and Human Development, Bethesda, Maryland
2 National Cancer Institute, Bethesda, Maryland
3 National Institute of Dental and Craniofacial Research, Bethesda, Maryland
Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, Kenneth M. Yamada (eds.)
Current Protocols in Cell Biology
Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.
DOI: 10.1002/0471143030.cbprefs00
Online Posting Date: May, 2001
Print Publication Date: October, 1998

Acknowledgments
This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the
hard work by the Current Protocols editorial staff at John Wiley and Sons. We are extremely grateful for the critical contributions by
Kathy Morgan (Series Editor) who kept the editors and the contributors on track and played a key role in bringing the entire project to
completion. Other skilled members of the Current Protocols staff who contributed to the project include Joseph White, Janet Blair,
Kathy Wisch, Michael Gates, Demetra Kagdis, Alice Ro, and Scott Holmes. The extensive copyediting required to produce an
accurate protocols manual was ably handled by Rebecca Barr, Allen Ranz, Elizabeth Harkins, Lisa Christenson, Connie Parks, Karen
Hopkin, Monte Kendrick, and Cathy Lundmark, and electronic illustrations were prepared by Gae Xavier Studios.

About Wiley InterScience | About Wiley | Privacy | Terms & Conditions
Copyright© 1999-2005 John Wiley & Sons, Inc. All rights reserved.
Recommended Background Reading
Preface
Juan S. Bonifacino1, Mary Dasso1, Joe B. Harford2, Jennifer Lippincott-Schwartz1, Kenneth M. Yamada3
1 National Institute of Child Health and Human Development, Bethesda, Maryland
2 National Cancer Institute, Bethesda, Maryland
3 National Institute of Dental and Craniofacial Research, Bethesda, Maryland
Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, Kenneth M. Yamada (eds.)
Current Protocols in Cell Biology
Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.
DOI: 10.1002/0471143030.cbprefs00
Online Posting Date: May, 2001
Print Publication Date: October, 1998

Recommended Background Reading
Alberts, B., Bray, D., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. 1997. Essential Cell Biology. Garland
Publishing, New York.
A basic introductory cell biology text written by the authors of Molecular Biology of the Cell.
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. 1994. Molecular Biology of the Cell, 3rd ed. Garland
Publishing, New York.
Darnell, J., Lodish, H., and Baltimore, D. 1995. Molecular Cell Biology. Scientific American Books, New York.
Two comprehensive and lucid textbooks that convey effectively the synergistic convergence of biochemistry, genetics, structural
biology, and traditional cell biology to form modern molecular and cell biology.
Juan S. Bonifacino, Mary Dasso, Joe B. Harford,
Jennifer Lippincott-Schwartz, and Kenneth M. Yamada

About Wiley InterScience | About Wiley | Privacy | Terms & Conditions
Copyright© 1999-2005 John Wiley & Sons, Inc. All rights reserved.
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Current Protocols in Cell Biology
Copyright ©2004 by John Wiley & Sons, Inc. All Rights Reserved.
Table of Contents | CP Titles

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Foreword HTML
Preface HTML
Chapter 1 Cell Culture
Introduction HTML PDF (Size 70KB)
Unit 1.1 Basic Techniques for Mammalian Cell Tissue Culture HTML PDF (Size 170KB)
Unit 1.2 Media for Culture of Mammalian Cells HTML PDF (Size 224KB)
Unit 1.3 Aseptic Technique for Cell Culture HTML PDF (Size 172KB)
Unit 1.4 Sterilization and Filtration HTML PDF (Size 230KB)
Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures HTML PDF (Size 463KB)
Unit 1.6 Media and Culture of Yeast HTML PDF (Size 176KB)
Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging HTML PDF (Size 332KB)
Chapter 2 Preparation and Isolation of Cells
Introduction HTML PDF (Size 63KB)
Unit 2.1 Establishment of Fibroblast Cultures HTML PDF (Size 706KB)
Unit 2.2 Preparation and Culture of Human Lymphocytes HTML PDF (Size 165KB)
Unit 2.3 Preparation of Endothelial Cells HTML PDF (Size 270KB)
Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation HTML PDF (Size
100KB)
Unit 2.5 Laser Capture Microdissection HTML PDF (Size 298KB)
Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures HTML PDF (Size 260KB)
Chapter 3 Subcellular Fractionation and Isolation of Organelles
Introduction HTML PDF (Size 34KB)
Unit 3.1 Overview of Cell Fractionation HTML PDF (Size 193KB)
Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains HTML PDF (Size 349KB)
Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation HTML PDF (Size 187KB)
Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation HTML PDF (Size 269KB)
Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
Centrifugation HTML PDF (Size 206KB)
Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
Centrifugation HTML PDF (Size 214KB)
Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae HTML PDF (Size
273KB)
Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae HTML PDF (Size 503KB)
Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
Centrifugation HTML PDF (Size 221KB)
Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues HTML PDF (Size 174KB)
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Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes HTML PDF (Size 390KB)
Chapter 4 Microscopy
Introduction HTML PDF (Size 87KB)
Unit 4.1 Proper Alignment and Adjustment of the Light Microscope HTML PDF (Size 929KB)
Unit 4.2 Fluorescence Microscopy HTML PDF (Size 178KB)
Unit 4.3 Immunofluorescence Staining HTML PDF (Size 228KB)
Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria HTML PDF (Size 503KB)
Unit 4.5 Basic Confocal Microscopy HTML PDF (Size 358KB)
Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues HTML PDF (Size 439KB)
Unit 4.7 Cryo-Immunogold Electron Microscopy HTML PDF (Size 497KB)
Unit 4.8 Correlative Video Light/Electron Microscopy HTML PDF (Size 407KB)
Unit 4.9 Polarization Microscopy HTML PDF (Size 1367KB)
Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells HTML PDF (Size 562KB)
Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues HTML PDF (Size 1421KB)
Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
Events HTML PDF (Size 430KB)
Unit 4.13 Fluorescent Labeling of Yeast HTML PDF (Size 524KB)
Unit 4A Organelle Atlas: Appendix to Chapter 4 HTML PDF (Size 2384KB)
Chapter 5 Characterization of Cellular Proteins
Introduction HTML PDF (Size 75KB)
Unit 5.1 Overview of the Physical State of Proteins Within Cells HTML PDF (Size 187KB)
Unit 5.2 Determining the Topology of an Integral Membrane Protein HTML PDF (Size 337KB)
Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density
Gradients HTML PDF (Size 311KB)
Unit 5.4 Analysis of the Association of Proteins with Membranes HTML PDF (Size 230KB)
Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration) HTML PDF (Size 233KB)
Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
Spectrometry HTML PDF (Size 308KB)
Chapter 6 Electrophoresis and Immunoblotting
Introduction HTML PDF (Size 48KB)
Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins HTML PDF (Size 404KB)
Unit 6.2 Immunoblotting and Immunodetection HTML PDF (Size 245KB)
Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots HTML PDF (Size 134KB)
Unit 6.4 Two-Dimensional Gel Electrophoresis HTML PDF (Size 290KB)
Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions HTML PDF (Size 208KB)
Unit 6.6 Staining Proteins in Gels HTML PDF (Size 209KB)
Unit 6.7 Agarose Gel Electrophoresis of Proteins HTML PDF (Size 187KB)
Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots HTML PDF (Size 218KB)
Unit 6.9 Digital Electrophoresis Analysis HTML PDF (Size 262KB)
Chapter 7 Protein Labeling and Immunoprecipitation
Introduction HTML PDF (Size 52KB)
Unit 7.1 Metabolic Labeling with Amino Acids HTML PDF (Size 147KB)
Unit 7.2 Immunoprecipitation HTML PDF (Size 271KB)
Unit 7.3 Metabolic Labeling with Sulfate HTML PDF (Size 98KB)
Unit 7.4 Metabolic Labeling with Fatty Acids HTML PDF (Size 175KB)
Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups HTML PDF (Size 143KB)
Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins HTML PDF (Size 111KB)
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Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins HTML PDF (Size 91KB)
Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars HTML PDF (Size 149KB)
Unit 7.9 Analysis of Oxidative Modification of Proteins HTML PDF (Size 239KB)
Unit 7.10 Radioiodination of Cellular Proteins HTML PDF (Size 156KB)
Chapter 8 Cell Cycle Analysis
Introduction HTML PDF (Size 40KB)
Unit 8.1 Overview of the Cell Cycle HTML PDF (Size 153KB)
Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle HTML PDF (Size 168KB)
Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle HTML PDF (Size 213KB)
Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry HTML PDF (Size 278KB)
Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells HTML PDF (Size 246KB)
Chapter 9 Cell Adhesion
Introduction HTML PDF (Size 149KB)
Unit 9.1 Cell-Substrate Adhesion Assays HTML PDF (Size 139KB)
Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force HTML PDF (Size 183KB)
Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion HTML PDF (Size 161KB)
Unit 9.4 Analyzing Integrin-Dependent Adhesion HTML PDF (Size 273KB)
Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules HTML PDF (Size 819KB)
Unit 9.6 Measurement of Adhesion Under Flow Conditions HTML PDF (Size 241KB)
Chapter 10 Extracellular Matrix
Introduction HTML PDF (Size 97KB)
Unit 10.1 Overview of Extracellular Matrix HTML PDF (Size 278KB)
Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors HTML PDF (Size 112KB)
Unit 10.3 Preparation of Gelled Substrates HTML PDF (Size 142KB)
Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9 Endodermal
Cells HTML PDF (Size 282KB)
Unit 10.5 Purification of Fibronectin HTML PDF (Size 131KB)
Unit 10.6 Purification of Vitronectin HTML PDF (Size 90KB)
Unit 10.7 Proteoglycan Isolation and Analysis HTML PDF (Size 166KB)
Unit 10.8 Matrix Metalloproteinases HTML PDF (Size 836KB)
Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured Fibroblasts HTML PDF (Size 388KB)
Unit 10.10 Purification and Analysis of Thrombospondin-1 HTML PDF (Size 223KB)
Unit 10.11 Purification of SPARC/Osteonectin HTML PDF (Size 266KB)
Chapter 11 In Vitro Reconstitution
Introduction HTML PDF (Size 87KB)
Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems HTML PDF (Size 148KB)
Unit 11.2 In Vitro Translation HTML PDF (Size 206KB)
Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells HTML PDF (Size 275KB)
Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes HTML PDF (Size 197KB)
Unit 11.5 In Vitro Analysis of SV40 DNA Replication HTML PDF (Size 164KB)
Unit 11.6 In Vitro Transcription HTML PDF (Size 211KB)
Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells HTML PDF (Size 439KB)
Unit 11.8 In Vitro Translation Using HeLa Extract HTML PDF (Size 144KB)
Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems HTML PDF (Size 292KB)
Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts HTML PDF (Size 332KB)
Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts HTML PDF (Size 215KB)
Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts HTML PDF (Size 155KB)
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