Proteins were expressed individually in Sf21 cells and whole cell extracts were mixed.
Interaction in vitro; bait produced as a recombinant fusion protein in baculovirus-infected Sf21 cell system; prey produced as recombinant fusion protein in baculovirus-infected Sf21 cell system.
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.
Factors were detected on polytene chromosomes using antibodies (to proteins) or biotinylated antisense probes (to ncRNA). The PLA signal develops only if probes to the two factors are in close proximity to each other (within tens of nanometers).
Source was larval salivary gland of wild-type fly line; proteins produced from endogenous gene.