Generation and verification of tagged protein arrays. (A) To tag ORFX as bait (V5–6×HIS) and prey (V5–3×VSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX. PCR products generated from the bait and prey templates are transformed into a- and α-cells, respectively. Homologous recombination occurs between the variable portion of the 5′ primer (light blue) and the 3′ terminus of the ORF, and between the variable portion of the 3′ primer (red) and the 3′ UTR) of ORFX. Transformants are selected on G418 plates, and colony PCR is performed to verify integration of the Kan^r downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kan^r, kanamycin resistance; loxp, site for CRE specific homologous recombination. (B) Monitoring the quality of the arrays by Western analysis of 14 ORFs as baits and preys. We chose proteins with a high level of expression (>2000 molecules per cell) as judged by Ghaemmaghami et al. (2003). The asterisk (*) denotes possible misloading or protein degradation. Note in the RAD51 lane the multiple protein products. Expected protein sizes are listed in Supplemental Table 1. (C) Analysis of effects on cell growth by tagging essential genes. A total of 24 strains with essential genes tagged as baits (6×HIS) and preys (3×VSV) were grown to saturation and spotted in 10-fold dilutions on YPD. Pictures were taken after 2 d at 30°C.