Proteins rarely act alone. The biological complexity of any cellular process—from signal transduction to immune response—depends on networks of interacting proteins. Understanding these interactions is foundational in cell biology, pharmacology, and systems biology.

Protein-Protein Interaction (PPI) assays allow researchers to map these complex networks, uncover interaction strengths, determine dynamic relationships, and validate therapeutic targets. This article explores the major classes of PPI assays, highlighting their principles, protocols, and real-world applications.

PPIs refer to the physical contacts between two or more protein molecules. These can be :

• Transient vs. Stable
• Obligate vs. Non-obligate
• Direct vs. Indirect

Mapping these interactions gives insights into cellular architecture, disease mechanisms, and potential intervention points.

🔹 Co-Immunoprecipitation (Co-IP)

• Principle: Uses antibodies to pull down a target protein and its binding partners.
• Application: Gold standard for validating PPIs under near-physiological conditions.
• Limitation: Cannot distinguish direct from indirect interactions.

🔹 Pull-Down Assays

• GST/His-tag fusion proteins are used to “pull down” interactors.
• Ideal for binary interaction validation.
• False positives possible due to overexpression or non-specific binding.

🔹 Surface Plasmon Resonance (SPR)

• Label-free detection of interaction kinetics (ka, kd, KD).
• High sensitivity.
• Requires immobilization of one protein on a sensor chip.

🔹 Isothermal Titration Calorimetry (ITC)

• Measures heat change during binding.
• Gives thermodynamic parameters (ΔH, ΔS, KD).
• Needs large protein quantities and purity.

🔹 Yeast Two-Hybrid (Y2H)

• Detects PPIs in yeast nucleus.
• Uses transcriptional activation as readout.
• Ideal for large-scale screening.

🔹 Bimolecular Fluorescence Complementation (BiFC)

• Fluorescent protein halves fuse to proteins of interest.
• Fluorescence occurs when proteins interact.
• Offers subcellular localization.

🔹 Förster Resonance Energy Transfer (FRET)

• Measures energy transfer between two fluorophores.
• Nanoscale proximity detection.
• Ideal for live-cell imaging.

🔹 Protein Microarrays

• Immobilized proteins on a slide probed with a partner protein.
• Suitable for PPI network screening.
• Requires large-scale protein expression.

🔹 Mass Spectrometry-Based Interactomics

• Affinity purification coupled to LC-MS/MS.
• Allows quantitative proteomics of complexes.
• Complex data analysis required.

🔹 Proximity Ligation Assay (PLA)

• In situ detection of PPIs using antibody-linked oligonucleotides.
• Amplification allows for single-molecule resolution.

🔹 Single-Molecule Pull-Down (SiMPull)

• Combines Co-IP and TIRF microscopy.
• Quantifies PPIs at single-molecule level.

🔹 Bioluminescence Resonance Energy Transfer (BRET)

• Uses luciferase donor and fluorescent acceptor.
• Ideal for live-cell PPI detection with high signal-to-noise.

Protein-protein interaction assays are central to understanding cellular systems, designing targeted therapeutics, and unraveling disease mechanisms. Whether you're mapping networks or validating a single interaction, the choice of assay depends on your biological question, resources, and resolution needs. As technologies evolve, combining complementary methods will offer a more complete view of the interactome.