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GTPase Protocols

The Ras Superfamily

Series: Methods in Molecular Biology

by Ed Manser and Thomas Leung

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Main description:

In the last 10 years researchers have firmly established key roles for R- related GTPases in almost every aspect of cell biology. In the 1980s the pro- oncogene Ras itself was the focus of interest, though in the 1990s this shifted to the increasing variety of Ras-related proteins. In this new decade much yet needs to be done to establish the role for all the small GTPases now uncovered by the human genome project. In particular, these GTPases need to be und- stood in the appropriate biochemical and cellular contexts. In the process of trying to uncover the role of these versatile proteins, a variety of novel te- niques and methodologies has been developed. These now enable investi- tors to move easily within a diversity of fields ranging from structural studies to real-time in vivo analysis of a GTPase. In recognition of the need for access to key background methodologies, GTPase Protocols: The Ras Superfamily is devoted to techniques that are pr- ently widely used and that will continue to be the standard for researchers worldwide. Each chapter is aimed at supplying detailed methodologies to allow reproduction in any laboratory, while also providing the general pr- ciples on which the methods are based. Some of the techniques grouped in the first section apply broadly to small GTPases, whereas others in Part II are more applicable within each GTPase subfamily.

Contents:

Part I. General Protocols

The GTPase Cycle: How Dominant Inhibitory Mutants Block the Biological Functions of Small GTPases
Edward J. Manser

Preparation of GTPases for Structural and Biophysical Analysis
Susan J. M. Smith and Katrin Rittinger

Using cDNA-Representational Difference Analysis (cDNA-RDA) in Combination with Microarrays to Identify
Rac Regulated Genes
Arndt A. P. Schmitz, Robert Lucito, and Linda Van Aelst

Fluorescence Methods in the Study of Small GTP-Binding Proteins
Mohammad Reza Ahmadian, Alfred Wittinghofer, and Christian Herrmann

Part II. Specific Methods
Ras Family GTPases

Ras and Rac as Activators of Reactive Oxygen Species (ROS)
Herbert Archer and Dafna Bar-Sagi

Interfering with Ras Signaling Using Membrane-Permeable Peptides or Drugs
Hiroshi Maruta, Hong He, and Thao Nheu

A Ras-Based Module to Generate 32P-Labeled Fusion Proteins for Blot Overlays
Zhoushen Zhao and Louis Lim

Rho Family GTPases

Determination of the Activity of Rho-Like GTPases in Cells
Angeliki Malliri, Jean P. ten Klooster, Cristina Olivo, and John G. Collard

Introduction of Dominant Inhibitory Proteins Directed Against ROK and MRCK Kinases
Ivan Tan, Louis Lim, and Thomas Leung

Effects of Rho Family GTPases on Cell-Cell Adhesion
Masaki Fukata, Masato Nakagawa, Shinya Kuroda, and Kozo Kaibuchi

Cell Motility and Invasion Assays
Jackie Banyard and Marc Symons

Rab Family GTPases

Isolation of Regulatory Proteins for the Rab3 Subfamily GTPases
Hiroyuki Nakanishi, Takuya Sasaki, and Yoshimi Takai

Analysis and Preparation of Stable Complexes between Rab GTPases
Rab Escort Protein, and Rab Geranylgeranyl Transferase
Jose B. Pereira-Leal, Anita Q. Gomes, and Miguel C. Seabra

Arf Family GTPases

Preparation ofMyristoylated Arf1 and Arf6 Proteins
Paul A. Randazzo and Henry M. Fales

ARF-Directed Guanine-Nucleotide-Exchange (GEP) Proteins
Gustavo Pacheco-Rodriguez, Joel Moss, and Martha Vaughan

Arf6 and its Role in Cytoskeletal Modulation
Julie G. Donaldson

ARF GTPase-Activating Protein 1
Irit Huber, Edna Cukierman, Miriam Rotman, and Dan Cassel

Ran GTPases

The Use of Permeabilized Cell Systems to Study Nuclear Transport
Amy M. Brownawell, James M. Holaska, Ian G. Macara, and Bryce M. Paschal

Analysis of Nuclear Protein Import and Export In Vitro Using Fluorescent Cargoes
Ralph H. Kehlenbach and Larry Gerace

Characterization of the Effects of RanGTP on the Microtubule Cytoskeleton
Sofia B. Lizarraga, Yixian Zheng, and Andrew R. Wilde

Index

PRODUCT DETAILS

ISBN-13: 9781617372612
Publisher: Springer (Humana Press Inc.)
Publication date: November, 2010
Pages: 280
Weight: 415g
Availability: Available
Subcategories: Biochemistry

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