Proteins and Proteomics

  1. Analysis of Protein Interactions
    1. Yeast Two-Hybrid (Y2H) System
      1. Principle and Workflow
        1. Transcriptional Activation
          1. Bait and Prey Constructs
            1. Reporter Gene Systems
            2. Variations and Improvements
              1. Membrane Y2H
                1. Reverse Y2H
                  1. Three-Hybrid Systems
                  2. Applications and Limitations
                    1. Interaction Screening
                      1. False Positives and Negatives
                        1. Protein Folding Requirements
                      2. Affinity Purification-Mass Spectrometry (AP-MS)
                        1. Tagging Strategies
                          1. Epitope Tags
                            1. Protein Tags
                              1. Endogenous Protein Purification
                              2. Complex Isolation
                                1. Purification Protocols
                                  1. Washing Stringency
                                    1. Elution Methods
                                    2. MS Analysis of Interactors
                                      1. Quantitative AP-MS
                                        1. Background Subtraction
                                          1. Statistical Analysis
                                          2. Computational Analysis
                                            1. Interaction Scoring
                                              1. Network Construction
                                            2. Co-immunoprecipitation (Co-IP)
                                              1. Antibody Selection
                                                1. Specificity Requirements
                                                  1. Cross-Reactivity Issues
                                                  2. Detection of Interacting Proteins
                                                    1. Western Blot Analysis
                                                      1. Mass Spectrometry Detection
                                                      2. Controls and Validation
                                                        1. Negative Controls
                                                          1. Reciprocal Co-IP
                                                        2. Proximity Ligation Assay (PLA)
                                                          1. Principle and Detection
                                                            1. Antibody Proximity
                                                              1. DNA Amplification
                                                                1. Fluorescent Detection
                                                                2. In Situ Applications
                                                                  1. Subcellular Localization
                                                                    1. Tissue Analysis
                                                                  2. Surface Plasmon Resonance (SPR)
                                                                    1. Real-Time Interaction Analysis
                                                                      1. Binding Kinetics
                                                                        1. Equilibrium Analysis
                                                                        2. Kinetic and Affinity Measurements
                                                                          1. Association and Dissociation Rates
                                                                            1. Equilibrium Dissociation Constants
                                                                            2. Experimental Design
                                                                              1. Surface Chemistry
                                                                                1. Analyte Preparation
                                                                                  1. Data Analysis
                                                                                2. Additional Interaction Methods
                                                                                  1. Fluorescence Resonance Energy Transfer (FRET)
                                                                                    1. Förster Distance
                                                                                      1. Donor-Acceptor Pairs
                                                                                        1. Live Cell Applications
                                                                                        2. Cross-linking Mass Spectrometry
                                                                                          1. Chemical Cross-Linkers
                                                                                            1. Photo-Cross-Linking
                                                                                              1. Structural Constraints
                                                                                              2. Protein Fragment Complementation Assays
                                                                                                1. Split Reporter Systems
                                                                                                  1. Bimolecular Fluorescence Complementation
                                                                                                  2. Thermal Shift Assays
                                                                                                    1. Protein Stability Changes
                                                                                                      1. Drug-Target Interactions

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                                                                                                  9. Quantitative Proteomics

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                                                                                                  11. Proteomic Data Analysis and Bioinformatics