Proteins and Proteomics

10.

Analysis of Protein Interactions

10.1.

Yeast Two-Hybrid (Y2H) System

10.1.1.

Principle and Workflow

10.1.1.1.

Transcriptional Activation

10.1.1.2.

Bait and Prey Constructs

10.1.1.3.

Reporter Gene Systems

10.1.2.

Variations and Improvements

10.1.2.1.

10.1.2.2.

10.1.2.3.

Three-Hybrid Systems

10.1.3.

Applications and Limitations

10.1.3.1.

Interaction Screening

10.1.3.2.

False Positives and Negatives

10.1.3.3.

Protein Folding Requirements

10.2.

Affinity Purification-Mass Spectrometry (AP-MS)

10.2.1.

Tagging Strategies

10.2.1.1.

10.2.1.2.

10.2.1.3.

Endogenous Protein Purification

10.2.2.

Complex Isolation

10.2.2.1.

Purification Protocols

10.2.2.2.

Washing Stringency

10.2.2.3.

Elution Methods

10.2.3.

MS Analysis of Interactors

10.2.3.1.

Quantitative AP-MS

10.2.3.2.

Background Subtraction

10.2.3.3.

Statistical Analysis

10.2.4.

Computational Analysis

10.2.4.1.

Interaction Scoring

10.2.4.2.

Network Construction

10.3.

Co-immunoprecipitation (Co-IP)

10.3.1.

Antibody Selection

10.3.1.1.

Specificity Requirements

10.3.1.2.

Cross-Reactivity Issues

10.3.2.

Detection of Interacting Proteins

10.3.2.1.

Western Blot Analysis

10.3.2.2.

Mass Spectrometry Detection

10.3.3.

Controls and Validation

10.3.3.1.

Negative Controls

10.3.3.2.

Reciprocal Co-IP

10.4.

Proximity Ligation Assay (PLA)

10.4.1.

Principle and Detection

10.4.1.1.

Antibody Proximity

10.4.1.2.

DNA Amplification

10.4.1.3.

Fluorescent Detection

10.4.2.

In Situ Applications

10.4.2.1.

Subcellular Localization

10.4.2.2.

Tissue Analysis

10.5.

Surface Plasmon Resonance (SPR)

10.5.1.

Real-Time Interaction Analysis

10.5.1.1.

Binding Kinetics

10.5.1.2.

Equilibrium Analysis

10.5.2.

Kinetic and Affinity Measurements

10.5.2.1.

Association and Dissociation Rates

10.5.2.2.

Equilibrium Dissociation Constants

10.5.3.

Experimental Design

10.5.3.1.

Surface Chemistry

10.5.3.2.

Analyte Preparation

10.5.3.3.

10.6.

Additional Interaction Methods

10.6.1.

Fluorescence Resonance Energy Transfer (FRET)

10.6.1.1.

Förster Distance

10.6.1.2.

Donor-Acceptor Pairs

10.6.1.3.

Live Cell Applications

10.6.2.

Cross-linking Mass Spectrometry

10.6.2.1.

Chemical Cross-Linkers

10.6.2.2.

Photo-Cross-Linking

10.6.2.3.

Structural Constraints

10.6.3.

Protein Fragment Complementation Assays

10.6.3.1.

Split Reporter Systems

10.6.3.2.

Bimolecular Fluorescence Complementation

10.6.4.

Thermal Shift Assays

10.6.4.1.

Protein Stability Changes

10.6.4.2.

Drug-Target Interactions

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9. Quantitative Proteomics

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11. Proteomic Data Analysis and Bioinformatics