Superdex 200 elution profile and SDS-PAGE analysis of serial chromatography fractionation of HS.

(A) Inhibitory fractions eluted from Phenyl Sepharose HP were pooled, concentrated, and subjected to gel filtration on Superdex 200 equilibrated in PBS. The column was calibrated with the molecular weight marker kit for gel filtration chromatography (Sigma). Fractions were tested for their protein contents (optical density [OD] 280 nm, dashed line) and inhibitory activity (closed circles). (B) Inhibitory fractions were pooled after each step and 10 μg protein aliquots were loaded per lane on 10% acrylamide gel: (a) HS; (b) breakthrough of Blue-Sepharose; (c) pool of inhibitory fractions from Q Sepharose; (d) pool of inhibitory fractions from Phenyl Sepharose HP; and (e) pool of inhibitory fractions from Superdex 200; the gel was stained with Coomassie blue.