Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).
Two-hybrid system: yeast GAL4-BD/GAL4-AD
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from S2 cell extract.
The N-terminal amphipathic helix of Arf protein was replaced with 10-His residues. His-tagged bait Arf protein was then used to coat the surface of liposomes containing low levels of a lipid with Ni-nitrilotriacetic acid (NTA) head group. Bait-coated liposomes are mixed with cell lysate, then liposomes are purified by centrifugation. This allows for the identification of protein interactions that occur preferentially or exclusively at the surface of a lipid bilayer.