Proteins rarely act alone. Detecting, validating and mapping their interactions — partners in a complex, relays of a signalling pathway, transient assemblies — is at the heart of mechanistic biology. Complementary methods — biochemical, genetic and imaging-based — make it possible to move from an interaction hypothesis to a proof.
Principle and workflow
Co-immunoprecipitation (co-IP) captures a target protein using its antibody bound to beads, then identifies the co-precipitated partners by immunoblot (known partner) or by mass spectrometry (unknown partner). Pull-down relies on a purified or tagged bait protein (His, GST, GFP-trap). The yeast two-hybrid (Y2H) detects binary interactions in vivo. PLA and FRET/FLIM, for their part, visualise a molecular proximity in situ.
Variants and options
Depending on the question: endogenous co-IP (native complexes), pull-down (direct interaction), AP-MS (large-scale interactome), Y2H (binary screening), PLA (in situ, on cells or tissues), FRET/FLIM (dynamics), BLI or SPR (affinity and kinetics). These approaches combine: a partner suggested by Y2H or AP-MS benefits from confirmation by co-IP, PLA or affinity measurement.
When and why these techniques
These methods come into play whenever an interaction hypothesis must be established or refuted: identifying a protein’s partners, validating a suspected complex, measuring an affinity, or locating an interaction within the cell. Y2H reveals binary interactions, including transient ones; AP-MS reports more stable co-complexes without giving their internal topology; co-IP confirms an association in its cellular context.
Each method has its blind spot. Co-IP loses weak or transient interactions during lysis and washing, and does not distinguish direct from indirect; Y2H is prone to both false positives and false negatives, operates outside the endogenous context and often misses interactions dependent on a post-translational modification; AP-MS generates abundance-related false positives; any fusion tag may disturb the binding site. Hence the rule: validate an interaction by at least one orthogonal method.
Inovarion’s expertise
Inovarion dissects protein interactions, from biochemical proof to in situ validation. Its publications attest to this: yeast two-hybrid, His-tag pull-down, GFP-trap and PLA uncovered the direct SLX4-RTEL1 interaction, which protects DNA replication; co-IP (GFP-trap) and FRET-FLIM established the assembly of the FcγRI receptor with the Syk kinase on endosomal signalling platforms, essential to antibody-dependent cytotoxicity; and co-immunoprecipitation coupled with mass spectrometry revealed that coagulation factor X circulates in complex with pentraxin-2. The methods chosen, and their cross-checking, respond to the nature of the interaction and its context.
See also: Proteomics & mass spectrometry ; Imaging & quantitative microscopy ; CRISPR & functional genomics.
Key publications
- Takedachi et al. SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations. Nature Structural & Molecular Biology, 2020. Record → · PubMed
- Benadda et al. Activating FcγR function depends on endosomal-signaling platforms. iScience, 2023. Record → · PubMed
- Muczynski et al. Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions. Blood, 2017. Record → · PubMed