Interaction inferred from the presence of two or more protein subunits in a partially purified protein preparation. If co-fractionation is demonstrated between 3 or more proteins, then add them as a complex.

An interaction is inferred when a bait-enzyme fusion protein selectively modifies a vicinal protein with a diffusible reactive product, followed by affinity capture of the modified protein and identification by mass spectrometric methods.

An interaction is inferred between proteins in vitro. This can include proteins in recombinant form or proteins isolated directly from cells with recombinant or purified bait. For example, GST pull-down assays where a GST-tagged protein is first isolated and then used to fish interactors from cell lysates are considered reconstituted complexes (e.g. PUBMED: 14657240, Fig. 4A or PUBMED: 14761940, Fig. 5). This can also include gel-shifts, surface plasmon resonance, isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI) experiments. The bait-hit directionality may not be clear for 2 interacting proteins. In these cases the directionality is up to the discretion of the curator.