Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).
Two-hybrid system: yeast GAL4-BD/GAL4-AD
Source was cross-linked nuclear extract of S2 cell line; bait produced from transfected construct; prey produced from endogenous gene.
Crude nuclear extracts were cross-linked using formaldehyde, sonicated, and subjected to ProteinA affinity purification with rabbit IgG agarose beads eluted under denaturing conditions, and subsequently using streptavidin agarose beads for Bio affinity purification. Purified peptides were eluted by on-bead trypsin digestion and mass spectrometric analysis.
Enrichment of prey protein was compared to input, mock and unrelated BioTAP affinity purifications using a statistical method (Bamse) developed by the authors to control for multiple sources of bias.