Bait fusion protein bound to lac0 sites and was visualized on polytene chromosomes. The ability of this chimera to recruit prey protein to lac0 sites was taken as evidence of direct physical interaction.
Source was larvae of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.
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.
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial or baculoviral system; prey produced as a recombinant fusion protein in bacterial or baculoviral system.
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial or baculoviral system; prey produced as a recombinant fusion protein in bacterial or baculoviral system.
