Figure 2

DEAE-Sephacel and PEDF-affinity column chromatography of media conditioned by retinoblastoma cells. GAGs and polyanions of CM were fractionated by anion-exchange column chromatography (CM^a). CM (2.5 mg protein) was applied to a DEAE-Sephacel column and the bound components were eluted with a linear gradient of 0.2–1 M NaCl. Panel A. Protein profile of the DEAE-Sephacel chromatography. Protein (--) and NaCl (-----) concentrations of eluted fractions are indicated. The load, unbound material (FT), and fractions 7 and 9 were resolved by 4–12% polyacrylamide gel electrophoresis under reducing conditions in two duplicate gels. Panels B and C. SDS-PAGE analysis of the DEAE chromatography. Gels stained with Coomassie Brilliant Blue (panel B) and with Toluidine Blue-O (panel C) are shown. Lane 1, load; lane 2, flow-through; lane 3 fraction #7; and lane 4, fraction #9. Panels D and E. PEDF-affinity column chromatography. CM^a was subjected to PEDF-affinity column chromatography. Bound GAGs were eluted with 3 M NaCl (CM^PEDF). Unbound material is FT and washes W₁ and W₂., A dot-blot stained with Toluidine Blue-O of fractions (as indicated) is shown in panel D. A 10–20% polyacrylamide gel (SDS-tricine) stained with Toluidine Blue-O is shown in Panel E with: lane 1, CM^PEDF; lane 2, 5 μg heparin of average MW 16,000; and lane 3, 5 μg HS. Migration positions of molecular-weight-standards are indicated to the right. Arrows indicate migration positions of GAGs from CM derived samples.