Two-Hybrid Systems: Methods and Protocols / Edition 1

by Paul N. MacDonald

Overview

The yeast two-hybrid system is one of the most widely used and productive techniques available for investigating the macromolecular interactions that affect virtually all biological processes. In Two-Hybrid Systems: Methods and Prools, Paul N. MacDonald has assembled a collection of these powerful molecular tools for examining and characterizing protein-protein, protein-DNA, and protein-RNA interactions. The techniques range from the most basic (introducing plasmids into yeasts, interaction assays, and recovering the plasmids from yeast) to the most advanced alternative strategies (involving one-hybrid, split two-hybrid, three-hybrid, membrane recruitment systems, and mammalian systems). Methods are also provided for dealing with the well-known problems of artifacts and false positives and for identifying the interacting partners in important biological systems, including the Smad and nuclear receptor pathways. To ensure ready reproducibility and robust results, each technique is described in step-by-step detail by researchers who employ it regularly.

Comprehensive and highly practical, Two-Hybrid Systems: Methods and Prools not only reveals how the great variety of plasmid vectors and approaches may be optimally deployed, but also quickly empowers novices to establish two-hybrid systems in their laboratories, and experienced researchers to expand their repertoire of techniques.

Product Details

ISBN-13:	9780896038080
Publisher:	Springer-Verlag New York, LLC
Publication date:	05/17/2001
Series:	Methods in Molecular Biology , #177
Edition description:	2001
Pages:	336
Product dimensions:	5.98(w) x 9.02(h) x 0.03(d)

About the Author

MacDonald, Paul N.

Table of Contents

General Methods.- The Two-Hybrid System.- Growth and Maintenance of Yeast.- Media Formulations for Various Two-Hybrid Systems.- Yeast Two-Hybrid Vectors and Strains.- High-Efficiency Transformation of Plasmid DNA into Yeast.- Qualitative and Quantitative Assessment of Interactions.- Strategies for Rescuing Plasmid DNA from Yeast Two-Hybrid Colonies.- False Positives.- Two-Hybrid System and False Positives.- Confirming Yeast Two-Hybrid Protein Interactions Using In Vitro Glutathione-S-Transferase Pulldowns.- Two-Hybrid Interactions Confirmed by Coimmunoprecipitation of Epitope-Tagged Clones.- Applications.- Smad Interactors in Bone Morphogenetic Protein Signaling.- Protein Interactions Important in Eukaryotic Translation Initiation.- Steroid Receptor and Ligand-Dependent Interaction with Coactivator Proteins.- Interaction of Cellular Apoptosis Regulating Proteins with Adenovirus Anti-apoptosis Protein E1B-19K.- Alternative Strategies.- Mammalian Two-Hybrid Assays.- One-Hybrid Systems f hor Detecting Protein-DNA Interactions.- The Split-Hybrid System.- Three-Hybrid Screens.- Three-Hybrid Screens for RNA-Binding Proteins.- Membrane Recruitment Systems for Analysis of Protein-Protein Interactions.

Editorial Reviews

Reviewer: Stephen A. Adam, PhD (Northwestern University Feinberg School of Medicine)
Description: This collection of methods and protocols for two-hybrid analysis of protein interactions is a volume in the fine Methods in Molecular Biology series.
Purpose: The purpose is to introduce the yeast two-hybrid system to investigators who would like to incorporate this approach into their research. The utility of the technique makes this book worthwhile since all the information necessary to get started is included. Anyone should be able to start using the two-hybrid system in their lab using this book as a guide.
Audience: The book is written for all levels of expertise from student to senior investigator. Each chapter is written by an expert in that particular area.
Features: The book is divided into four main sections. The first is a general collection of methods that are sufficient to get anyone started on the technique. Section two covers the problem of dealing with false positives and strategies to confirm authentic interactions. The third section gives examples of the use of the two-hybrid system in biological systems and the fourth section describes various adaptations of the two-hybrid assay. The protocols are detailed and easy to follow and even the preparation of standard buffers is described. A great deal of information on basic yeast techniques is provided for investigators who have not previously worked with yeast. Each chapter contains numerous notes and insights on what to keep an eye out for and potential problems.
Assessment: This would be an excellent addition to any laboratory considering starting the two-hybrid system. The first chapter on the origins of the method is a must read for everyone, especially students.

Doody's Review Service

"The utility of the technique makes this book worthwhile since all the information necessary to get started is included. Anyone should be able to start using the two-hybrid system in their lab using this book as a guide. The book is written for all levels of expertise from student to senior investigator. Each chapter is written by an expert in that particular area. The prools are detailed and easy to follow and even the preparation of standard buffers is described. A great deal of information on basic yeast techniques is provided for investigators who have not previously worked with yeast. Each chapter contains numerous notes and insights on what to keep an eye out for and potential problems. This would be an excellent addition to any laboratory considering starting the two-hybrid system. The first chapter on the origins of the method is a must read for everyone, especially students."-98 Score, 5 stars, Doody's Health Science Book Review Journal

"The stated goal of the volume is "to introduce the yeast two-hybrid systems to students, research assistants, research associates, and other more senior investigators considering this as a new approach." This goal is well achieved as the volume is easy to read and comprehensive.... the first section of this volume provides in depth detailing of yeast prools, including several very useful trouble shooting solutions and tips well beyond the scope of those found in the instruction manuals accompanying commercial kits....this book is a worthwhile investment to ensure that the most is gained from your two-hybrid experiments and thus I would recommend it." - Molecular Biotechnology

"This current volume is a practical, laboratory manual with detailed methods and trouble-shooting tips for the yeast two-hybrid approach. The volume includes a brief historical overview, with chapters addressing yeast husbandry and media preparation and yeast genetic modification. The manual lso includes detailed accountsof how to construct a two-hybrid screen and how to analyse the positives. I found the book easy to read, and the prools were clear and straightforward to follow. There are a number of useful tables, including yeast genotypes and genetic markers as well as available vectors. I would recommend this book as a practical laboratory manual for anyone contemplating a search for a partner protein using the yeast two-hybrid approach." -The Endocrinologist

From the Publisher

Reviewer: Stephen A. Adam, PhD(Northwestern University Feinberg School of Medicine)
Description: This collection of methods and protocols for two-hybrid analysis of protein interactions is a volume in the fine Methods in Molecular Biology series.
Purpose: The purpose is to introduce the yeast two-hybrid system to investigators who would like to incorporate this approach into their research. The utility of the technique makes this book worthwhile since all the information necessary to get started is included. Anyone should be able to start using the two-hybrid system in their lab using this book as a guide.
Audience: The book is written for all levels of expertise from student to senior investigator. Each chapter is written by an expert in that particular area.
Features: The book is divided into four main sections. The first is a general collection of methods that are sufficient to get anyone started on the technique. Section two covers the problem of dealing with false positives and strategies to confirm authentic interactions. The third section gives examples of the use of the two-hybrid system in biological systems and the fourth section describes various adaptations of the two-hybrid assay. The protocols are detailed and easy to follow and even the preparation of standard buffers is described. A great deal of information on basic yeast techniques is provided for investigators who have not previously worked with yeast. Each chapter contains numerous notes and insights on what to keep an eye out for and potential problems.
Assessment: This would be an excellent addition to any laboratory considering starting the two-hybrid system. The first chapter on the origins of the method is a must read for everyone, especially students.

This collection outlines the molecular tools for examining and characterizing protein-protein, protein-DNA, and protein-RNA interactions. The techniques discussed range from the most basic (introducing plasmids into yeasts, for example) to the most complex (involving one-hybrid, split two-hybrid, three hybrid, membrane recruitment systems, and mammalian systems). Methods are also provided for dealing with common problems of artifacts and false positives and for identifying the interacting partners in biological systems, including the Smad and nuclear receptor pathways. Each technique is described in step-by-step detail. Plastic comb binding. Annotation c. Book News, Inc., Portland, OR (booknews.com)

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