Two-Hybrid Systems: Methods and Protocols

✍ Scribed by Paul N. MacDonald

• Publisher: Humana Press
• Year: 2001
• Tongue: English
• Leaves: 335
• Series: Methods in Molecular Biology 177
• Edition: 1
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Preface......Page 5
Contents......Page 9
PART I. GENERAL METHODS......Page 13
The Two-Hybrid System......Page 14
Growth and Maintenance of Yeast......Page 20
Media Formulations for Various Two-Hybrid Systems......Page 26
Yeast Two-Hybrid Vectors and Strains......Page 51
High-Efficiency Transformation of Plasmid DNA into Yeast......Page 93
Qualitative and Quantitative Assessment of Interactions......Page 106
Strategies for Rescuing Plasmid DNA from Yeast Two-Hybrid Colonies......Page 114
PART II. FALSE POSITIVES......Page 127
Two-Hybrid System and False Positives......Page 128
Confirming Yeast Two-Hybrid Protein Interactions Using In Vitro Glutathione-S-Transferase Pulldowns......Page 140
Two-Hybrid Interactions Confirmed by Coimmunoprecipitation of Epitope-Tagged Clones......Page 156
PART III. APPLICATIONS......Page 165
Smad Interactors in Bone Morphogenetic Protein Signaling......Page 166
Protein Interactions Important in Eukaryotic Translation Initiation......Page 182
Steroid Receptor and Ligand-Dependent Interaction with Coactivator Proteins......Page 202
Interaction of Cellular Apoptosis Regulating Proteins with Adenovirus Anti-apoptosis Protein E1B-19K......Page 213
PART IV. ALTERNATIVE STRATEGIES......Page 221
Mammalian Two-Hybrid Assays......Page 222
One-Hybrid Systems f hor Detecting Protein-DNA Interactions......Page 241
The Split-Hybrid System......Page 260
Three-Hybrid Screens......Page 270
Three-Hybrid Screens for RNA-Binding Proteins......Page 289
Membrane Recruitment Systems for Analysis of Protein–Protein Interactions......Page 317
Index......Page 327