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Concentrating on research methodologies, 20 chapters, presented by Manser and Leung (both of the Glaxo-IMCB Group, Institute of Molecular and Cell Biology, Singapore), look at ways to study a group of proteins that play major roles in cell biology. Step-by-step instructions are given in the realms of general protocols and specific methods. The general principles on which methods are based are discussed and common pitfalls are identified.Book News, Inc.

Book Info

Institute of Molecular and Cell Biology, Singapore. Chapters are aimed at supplying detailed methodologies to allow reproduction in any laboratory. Provides the general principles on which the methods are based. For researchers.

Book Description

Edward J. Manser and Thomas Leung have collected the key techniques currently in use to probe the function of these ubiquitous proteins both in vitro and in vivo. Presented in a format that ensures ready reproducibility by accomplished experimentalists who have refined the various methods in their laboratories, each technique includes step-by-step instructions, tips on avoiding pitfalls and troubleshooting, and ancillary notes explaining how to adapt each procedure in the event of problems. The methods cover the spectrum of core techniques required for the five major GTPase subfamilies (Ras, Rho, Rab, Arf, and Ran) and permit a diversity of applications ranging from structural studies on a GTPase to real time in vivo analysis. Timely and highly practical, GTPase Protocols: The Ras Superfamily illuminates the powerful techniques used by investigators today to study this special family of proteins that plays such important roles in human health and disease.

Preface
Contributors
Pt. I General Protocols
1 The GTPase Cycle: How Dominant Inhibitory Mutants Block the Biological Functions of Small GTPases 3
2 Preparation of GTPases for Structural and Biophysical Analysis 13
3 Using cDNA-Representational Difference Analysis (cDNA-RDA) in Combination with Microarrays to Identify Rac Regulated Genes 25
4 Fluorescence Methods in the Study of Small GTP-Binding Proteins 45
Pt. II Specific Methods: Ras Family GTPases
5 Ras and Rac as Activators of Reactive Oxygen Species (ROS) 67
6 Interfering with Ras Signaling Using Membrane-Permeable Peptides or Drugs 75
7 A Ras-Based Module to Generate [superscript 32]P-Labeled Fusion Proteins for Blot Overlays 87
Pt. II Specific Methods: Rho Family GTPases
8 Determination of the Activity of Rho-Like GTPases in Cells 99
9 Introduction of Dominant Inhibitory Proteins Directed Against ROK and MRCK Kinases 111
10 Effects of Rho Family GTPases on Cell-Cell Adhesion 121
11 Cell Motility and Invasion Assays 129
Pt. II Specific Methods: Rab Family GTPases
12 Isolation of Regulatory Proteins for the Rab3 Subfamily GTPases 143
13 Analysis and Preparation of Stable Complexes between Rab GTPases, Rab Escort Protein, and Rab Geranylgeranyl Transferase 157
Pt. II Specific Methods: Arf Family GTPases
14 Preparation of Myristoylated Arf1 and Arf6 Proteins 169
15 ARF-Directed Guanine-Nucleotide-Exchange (GEP) Proteins 181
16 Arf6 and its Role of Cytoskeletal Modulation 191
17 ARF GTPase-Activating Protein 1 199
Pt. II Specific Methods: Ran GTPases
18 The Use of Permeabilized Cell Systems to Study Nuclear Transport 209
19 Analysis of Nuclear Protein Import and Export In Vitro Using Fluorescent Cargoes 231
20 Characterization of the Effects of RanGTP on the Microtubule Cytoskeleton 247
Index 261