![]() 3) 4 Notes References Chapter 4: Detection of Protein-Protein Interactions Utilizing the Split-Ubiquitin Membrane-Based Yeast Two-Hybrid System 1 Introduction 2 Materials 3 Methods 3.1 Generation of NMY51 Yeast Strain Expressing the Functional Bait Protein 3.1.1 Transformation of the Bait Construct into NMY51 3.1.2 Bait Protein Expression Check by Western Blot 3.1.3 Bait Functional Assay 3.2 Standardization of Screening Stringency Using a Pilot Transformation 3.3 Library Screening and Confirmation of Interaction 3.3.1 Library Transformation 3.3.2 Recovery of Prey Plasmid 3.3.3 Bait-Dependency Test 3.3.4 Qualitative ß-Galactosidase Activity (Colony-Lift Filter Assay) 4 Notes References Chapter 5: Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in. 2.5 Yeast Selection Media for mbSUS, GPS, and Tri-SUS Systems 2.6 The Amino Acid and Nucleobase Stock Solutions for mbSUS, GPS, and Tri-SUS Systems (see Note 3) 3 Methods 3.1 Protocol for Yeast Transformation and Selection for mbSUS, GPS, and Tri-SUS Assays (See Note 4) 3.2 The Steps for the Protocol for Yeast Growth Assay (See Note 4 and Fig. 2) 3.2 Construction of the Bait-hSos Protein Fusion and Target Plasmid 3.3 Competent Cell Preparation for cdc25H Yeast Strain 3.4 Transformation of the Plasmids into Yeast and Identification of the Interaction System 3.5 Confirmation of the Interaction Between Bait Protein and Target Protein Using a Co-transformation Approach 3.6 Confirmation of the Interaction Between Bait Protein and Target Protein Using Yeast Mating System 4 Notes References Chapter 3: Analyzing Protein-Protein Interactions Using the Split-Ubiquitin System 1 Introduction 1.1 Yeast mbSUS Reporter System 1.2 mbSUS Assays for Analysis of Tripartite Interactions 2 Materials 2.1 Yeast SUS Assays Chemicals List 2.2 Antibodies 2.3 mbSUS Yeast Strains 2.4 The Reagents and Media for Yeast Growth and Transformation. ![]() Table of contents : Preface Contents Contributors Chapter 1: Yeast Two-Hybrid Technique to Identify Protein-Protein Interactions 1 Introduction 2 Materials 2.1 Yeast Two-Hybrid Library Construction 2.2 Constructs for Pairwise Y2H 2.3 Yeast Transformation 2.4 Y2H Library Screening 3 Methods 3.1 Yeast Two-Hybrid Library Construction 3.2 Yeast Transformation 3.3 Testing Autoactivation 3.4 Yeast Two-Hybrid Library Screening 3.5 Pairwise Y2H Screening 4 Notes References Chapter 2: Cytotrap: An Innovative Approach for Protein-Protein Interaction Studies for Cytoplasmic Proteins 1 Introduction 2 Materials 2.1 Reagents and Supply Items 2.2 Specialist Equipment 2.3 The Composition of All Buffers, Media, and Solutions 3 Methods 3.1 Processing the Yeast Strain and Glycerol Stock Preparation Process (Fig. ![]()
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