[Abstract:]
A collection of highly successful biochemical, biophysical, genetic, and computational techniques for studying protein?protein interactions. These readily reproducible methods demonstrate how to identify protein interaction partners, qualitatively or quantitatively measure protein-protein interactions in vitro or in vivo, monitor protein?protein interactions as they occur in living cells, and determine interaction interfaces. The techniques described utilize a variety of cutting-edge technologies, including surface plasmon resonance (SRP), fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), isothermal titration calorimetry (ITC), circular dichroism (CD), protein fragment complementation assays (PCA), various two-hybrid systems, and proteomics and bioinformatics-based approaches, such as the Scansite program for computational analysis. These powerful tools are essential for deciphering how proteins interact with each other to form biological networks, as well as for unraveling protein-protein interactions in disease to find novel therapeutic targets.
[目录]
1. Structural Basis of Protein?Protein Interactions
2. Quantitative Analysis of Protein?Protein Interactions
3. Characterization of Protein?Protein Interactions by Isothermal Titration Calorimetry
4. Circular Dichroism Analysis for Protein?Protein Interactions
5. Protein?Protein Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy
6. Measuring Rhodopsin?G-Protein Interactions by Surface Plasmon Resonance
7. Using Light Scattering to Determine the Stoichiometry of Protein Complexes
8. Sedimentation Equilibrium Studies
9. Analysis of Protein?Protein Interactions by Simulation of Small-Zone Gel Filtration Chromatography
10. Fluorescence Gel Retardation Assay to Detect Protein?Protein Interactions
11. Fluorescence Polarization Assay to Quantify Protein?Protein Interactions
12. Studying Protein?Protein Interactions via Blot Overlay or Far Western Blot
13. Glutathione-S-Transferase?Fusion Based Assays for Studying Protein?Protein Interactions
14. Affinity Capillary Electrophoresis Analyses of Protein?Protein Interactions in Target-Directed Drug Discovery
15. Mapping Protein?Ligand Interactions by Hydroxyl-Radical Protein Footprinting
16. Use of Phage Display and Polyvalency to Design Inhibitors of Protein?Protein Interactions
17. A Bacterial Two-Hybrid System Based on Transcription Activation
18. Using the Yeast Two-Hybrid System to Identify Interacting Proteins
19. Analysis of Protein?Protein Interactions Utilizing Dual Bait Yeast Two-Hybrid System
20. The Split-Ubiquitin Membrane-Based Yeast Two-Hybrid System
21. Reverse Two-Hybrid Techniques in the Yeast Saccharomyces cerevisiae
22. Mammalian Two-Hybrid Assay for Detecting Protein?Protein Interactions In Vivo
23. Co-Immunoprecipitation from Transfected Cells
24. Microscopic Analysis of Fluorescence Resonance Energy Transfer (FRET)
25. Monitoring Molecular Interactions in Living Cells Using Flow Cytometric Analysis of Fluorescence Resonance Energy Transfer
26. Fluorescence Correlation Spectroscopy: A New Tool for Quantification of Molecular Interactions
27. Confocal Microscopy for Intracellular Co-Localization of Proteins
28. Mapping Biochemical Networks With Protein-Fragment Complementation Assays
29. In Vivo Protein Cross-Linking
30. Computational Prediction of Protein?Protein Interactions
31. Affinity Methods for Phosphorylation-Dependent Interactions
32. Two-Dimensional Gel Electrophoresis for Analysis of Protein Complexes
33. Sample Preparation of Gel Electrophoretically Separated Protein Binding Partners for Analysis by Mass Spectrometry
34. Quantitative Protein Analysis by Solid Phase Isotope Tagging and Mass Spectrometry
35. Internet Resources for Studying Protein?Protein Interactions
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