Yeast two-hybrid screening

Yeast cells containing the GH bait plasmid and the prey library yeast cells were mated, and were plated on his-selective medium. Note that on this selective medium, only His-plus diploid cells can grow irrespective of the presence of the bait and prey plasmid. The His-plus colonies were observed under a microscope (BX61; Olympus Co.) for red and cyanic fluorescence, and only entirely red and cyanic colonies (i.e., colonies that grew on the selective plate dependently on both the bait and prey plasmids) were picked. The picked cells were successively streaked for three times on the same histidine-minus selective medium and also on the non-selective medium, containing histidine, to verify bait- and prey-dependency. The clones that went through the procedure were subjected to PCR amplification and sequence determination.