Figure 3.

Purification of high-molecular-weight transmembrane mucins from stratified human corneal epithelial cells. An established two-step isolation procedure using size-exclusion chromatography and isopycnic density centrifugation was employed to purify transmembrane mucins. a, ∼10 mg of cell extract was applied to a Sepharose CL-4B column (1 × 30 cm) and eluted with PBS, pH 7.5. Fractions were evaluated for glycoprotein content using PAS staining. The asterisk indicates the stacking gel. b, by Western blotting, the high-molecular-weight fractions F1-F4 contained MUC1, MUC4, and MUC16 but not the smaller MUC20 transmembrane mucin. c, these fractions were pooled and subjected to isopycnic density centrifugation. Fractions at a buoyant density range of 1.26–1.43 g/ml tested positive for MUC1, MUC4, and MUC16 and were combined for N-glycan sequencing analyses. Coomassie G-250 staining revealed the presence of some low-molecular-weight bands in the mucin isolate; however, these bands were not glycosylated, as shown by PAS staining.