Two-Hybrid Systems: Methods and Protocols

Author:   Paul N. MacDonald
Publisher:   Humana Press Inc.
Edition:   2001 ed.
Volume:   177
ISBN:  

9780896038325


Pages:   336
Publication Date:   17 May 2001
Format:   Hardback
Availability:   In Print   Availability explained
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Two-Hybrid Systems: Methods and Protocols


Overview

Paul N. MacDonald has assembled a collection of 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 problem 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.

Full Product Details

Author:   Paul N. MacDonald
Publisher:   Humana Press Inc.
Imprint:   Humana Press Inc.
Edition:   2001 ed.
Volume:   177
Dimensions:   Width: 15.50cm , Height: 2.00cm , Length: 23.50cm
Weight:   0.670kg
ISBN:  

9780896038325


ISBN 10:   0896038327
Pages:   336
Publication Date:   17 May 2001
Audience:   College/higher education ,  Professional and scholarly ,  Undergraduate ,  Professional & Vocational
Format:   Hardback
Publisher's Status:   Active
Availability:   In Print   Availability explained
This item will be ordered in for you from one of our suppliers. Upon receipt, we will promptly dispatch it out to you. For in store availability, please contact us.

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.

Reviews

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 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. 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 protocols, 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 accounts of how to construct a two-hybrid screen and how to analyse the positives. I found the book easy to read, and the protocols 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


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 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. 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 protocols, 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 accounts of how to construct a two-hybrid screen and how to analyse the positives. I found the book easy to read, and the protocols 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


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 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. 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 protocols, 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 accounts of how to construct a two-hybrid screen and how to analyse the positives. I found the book easy to read, and the protocols 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


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