Figure 1. Purification and identification of syndecan-3-binding protein.

(A) Syndecan-3-binding protein was fractionated by chromatography on Sepharose CL-4B and the elution profile and elution volume V_e were analysed. The void volume V_o and the maximum elution volume V_t were measured with respect to the elution positions of Blue Dextran and p-nitrophenol respectively. (B) Aliquots of each fraction were subjected to ligand overlay assay using biotinylated soluble syndecan-3. (C, D) Positive fractions [K_av=(V_e−V_o)/(V_t−V_o)=0.27–0.44] were collected, treated with (+) or without (–) chondroitinase ABC and then separated by SDS/PAGE (6% gel), followed by either visualization by CBB staining (C) or immunochemical detection using anti-neurocan monoclonal antibody 3A11 (D). EN, the same amount of chondroitinase ABC used in (C, D) was subjected to SDS/PAGE followed by the same procedure as described above.