Figure 1: Representative profiles from the four chromatographic steps for purifying the male-attracting substance from sexually developed red-bellied newt (Cynops pyrrhogaster) oviducts.

(a) Gel-filtration fast protein liquid chromatography (FPLC; Superose 12 HR 10/30) of the aqueous extract from 20 pieces of oviduct following partial purification on a Sep-Pak C₁₈ cartridge. The low-molecular-weight (M.W. < 3,000) fractions denoted by the bar contained the active substance and were pooled for further purification. (b) Reversed phase high-performance liquid chromatography (HPLC; Puresil C18) of fractions containing the active substance (20 pieces equivalent) that were obtained in (a). The fractions that possessed a male-attracting activity are indicated by the bar. (c) Reversed phase HPLC (Nucleosil 120 3C18) of the fraction (105 pieces equivalent) possessing male-attracting activity that was separated in (b). The fraction that possessed a male-attracting activity is indicated by an arrow. (d) Reversed phase HPLC (Inertsil Ph) of the fraction (190 pieces equivalent) containing the male-attracting substance that was separated in (c). The peak of the fraction that possessed the male-attracting activity is indicated by an arrow. The final product was estimated to be 40 ng/piece. See Supplementary Fig. S2 for results of the associated preference tests.