An interaction is inferred from the identification of two or more protein subunits in a purified protein complex, as obtained by classical biochemical fractionation or affinity purification and one or more additional fractionation steps.

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.

Bait protein expressed as a DNA binding domain (DBD) fusion and prey expressed as a transcriptional activation domain (TAD) fusion and interaction measured by reporter gene activation.