Figure 1.
Cross-and-Capture Assay. (A,B) A strain containing the bait ORFX tagged with a V5 epitope and six histidines (6×HIS) is crossed with strains that contain prey ORFY or ORFZ tagged with a V5 epitope followed by a triple VSV tag (3×VSV). Diploids, which express both tagged bait and prey, are grown on selective medium. Protein extracts from the diploids are then incubated with nickel beads (Ni2+-NTA), allowing isolation of bait (Protein X–6×HIS) and bait-associated prey protein (Protein Y–3×VSV) (A), whereas a non-interacting protein (Protein Z–3×VSV) will not bind (B). Proteins are then separated by SDS-PAGE, and blots are probed for bait and prey (anti-V5 antibody) and specifically for the prey (anti-VSV antibody) by immunoblot.