An interaction is inferred when a bait protein is affinity captured from cell extracts by either polyclonal antibody or epitope tag and the associated interaction partner is identified by mass spectrometric methods.

Interaction directly demonstrated at the atomic level by X-ray crystallography. Also used for NMR or Electron Microscopy (EM) structures. If there is no obvious bait-hit directionality to the interaction involving 3 or more proteins, then the co-crystallized proteins should be listed as a complex.

An interaction is detected between two proteins using chemically reactive or photo-activatable cross-linking reagents that covalently link amino acids in close proximity, followed by mass spectrometry analysis to identify the linked peptides (reviewed in PMID 37406423, 37104977). Experiments may be carried with live cells or cell lysates in which all proteins are expressed at endogenous levels (e.g. PMID 34349018, 35235311) or with recombinant proteins (e.g., PMID 28537071).

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.

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.