Fig. 2.

Chromatographic resolution of Siglec-8 ligands extracted from human airway. (A) Sephacryl S-500 column chromatography of Siglec-8 ligands extracted from human trachea. Elution of proteins (A₂₈₀, dashed line) and Siglec-8 binding activity (solid line) determined by semiquantitative dot blot Siglec-8-Fc overlay are plotted against the elution volume. (B) Composite gel electrophoresis of active binding fractions. Equal aliquots of fractions with Siglec-8-Fc-binding activity detected by semiquantitative dot blot were resolved by 1.5% acrylamide, 2% agarose composite gel electrophoresis, blotted to PVDF membranes and probed for Siglec-8-Fc binding. Images of three replicate gels (boxed) used to accommodate fractions across the active elution fractions were stitched together by lining up common molecular weight standards (Stds, left). Fractions were pooled to represent three size classes of Siglec-8 ligands (designated by estimated molecular weight in daltons): S8-1M, S8-600K and S8-250K.