Dictyostelium discoideum Dictyostelium [1–3] [4] Dictyostelium [5] [6] [7] [8,9] [10,11] [12–15] [16,17] Dictyostelium Materials and methods Construction of plasmid vectors Bgl Xho Bgl Xho Table 2 [11] Bam Hin [10] Cla Xho Hin Spe Eco Cla Table 2 Generation and transformation of construct p34-ArcCTAP Dictyostelium ArpE [5,19] ArpE Table 1 Bam Xba Bam Xba D. discoideum Purification of TAP-tagged protein complexes [10,11] 9 g 2 2 Protein mass fingerprinting 4 3 N [20] trans 4 2 4 http://www.ebi.ac.uk/swissprot/ Results and discussion Cloning strategies Dictyostelium [21] Dictyostelium [18] Fig. 1 Xba Hin Nhe Sal Bgl Xho Table 1 Fig. 1 Dictyostelium  Xho Bgl Eco Nhe Sal Table 2 Purification of the Dictyostelium Arp2/3 complex with TAP-tagged p34-Arc Dictyostelium D. discoideum [19] D. discoideum [14] D. discoideum Table 1 Bam Xba Bam Xba D. discoideum 9 Fig. 2 Dictyostelium [19] D. discoideum Fig. 2 Dictyostelium Conclusions Dictyostelium bsr Preferentially, the “bait” gene of choice should be expressed under its own promoter from a single copy vector in a “bait” null mutant. This will avoid artefacts due to ectopic expression and competition of the endogenous “bait” with the tagged “bait” for binding to cellular proteins. Moreover, rescue of the null mutant phenotype by the tagged “bait” will ensure that the tags are not interfering with protein function. However, it may often be desirable to overexpress the “bait” to obtain a sufficient yield of protein complexes, although this also increases the probability of pulling down unrelated proteins. Dictyostelium