GTPase Protocols

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.

Includes supplementary material: sn.pub/extras

Klappentext
Small GTPase binding proteins (GTPases) are an ancient group of proteins that play key roles in almost every aspect of cell biology, from cell proliferation to nuclear transport. In GTPase Protocols: The Ras Superfamily, 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.

Inhalt
General Protocols.- The GTPase Cycle How Dominant Inhibitory Mutants Block the Biological Functions of Small GTPases.- Preparation of GTPases for Structural and Biophysical Analysis.- Using cDNA-Representational Difference Analysis (cDNA-RDA) in Combination with Microarrays to Identify Rac Regulated Genes.- Fluorescence Methods in the Study of Small GTP-Binding Proteins.- Specific Methods.- Ras and Rac as Activators of Reactive Oxygen Species (ROS).- Interfering with Ras Signaling Using Membrane-Permeable Peptides or Drugs.- A Ras-Based Module to Generate 32P-Labeled Fusion Proteins for Blot Overlays.- Determination of the Activity of Rho-Like GTPases in Cells.- of Dominant Inhibitory Proteins Directed Against ROK and MRCK Kinases.- Effects of Rho Family GTPases on Cell-Cell Adhesion.- Cell Motility and Invasion Assays.- Isolation of Regulatory Proteins for the Rab3 Subfamily GTPases.- Analysis and Preparation of Stable Complexes between Rab GTPases, Rab Escort Protein, and Rab Geranylgeranyl Transferase.- Preparation of Myristoylated Arf1 and Arf6 Proteins.- ARF-Directed Guanine-Nucleotide-Exchange (GEP) Proteins.- Arf6 and its Role in Cytoskeletal Modulation.- ARF GTPase-Activating Protein 1.- The Use of Permeabilized Cell Systems to Study Nuclear Transport.- Analysis of Nuclear Protein Import and Export In Vitro Using Fluorescent Cargoes.- Characterization of the Effects of RanGTP on the Microtubule Cytoskeleton.