Fig. 2. Purification of the morphogenetic factor. (A) Silver-stained SDS-PAGE gel of active fractions from column chromatography of BSN-CM. Lane 1, whole BSN-CM; lane 2, active fraction from heparin sepharose column; lane 3, active fraction from the Resource phenyl sepharose hydrophobic interaction column. (B) Elution profile from the Resource S cation exchange column of the active fraction from Resource phenyl sepharose column. A single, sharp protein peak was eluted at 0.4-0.6 M NaCl. Each of the individual 1 ml fractions eluted from the column are indicated by the numbers (1-8) above the x axis. (C) Phase-contrast photomicrographs of isolated ureteric buds cultured for 7 days in the presence of each 1 ml fraction from the Resource S cation exchange column (1-8 in B) supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml FGF1. Fraction 4, which corresponded to the protein peak on the elution profile (B) exhibited potent morphogenetic activity. Scale Bar, 500 µm. (D) Silver-stained SDS-PAGE gel of each fraction (1-8) eluted from the Resource S cation exchange column (B). Fraction 4, which possessed potent morphogenetic activity (C) contained a single low molecular mass band, which was identified as pleiotrophin by mass spectrometry. (E) Immunoblot analysis of the individual fractions eluted from the Resource S cation exchange column (1-8 in B). The blot was probed with anti-pleiotrophin antibodies. Rh-PTN; 250 ng of human recombinant pleiotrophin as a positive control.
