FIG. 3.

Deglycosylation of the high-molecular-weight variant of LecB (HW LecB). The affinity-purified periplasmic LecB variants (A) and size exclusion chromatography-separated HW LecB (B) were exposed to N-glycosidase F from Flavobacterium meningosepticum (PNGase F; Roche). Amounts of 10 μg of purified periplasmic LecB and 100 ng of separated HW LecB were incubated with 2 U of PNGase F or without any enzyme in 50 mM sodium-phosphate buffer (pH 7.5) in a final volume of 10 μl for 16 h at 37°C. Lanes: 1, molecular weight marker (in thousands); 2 and 4, periplasmic LecB (2) and HW LecB (4) without the addition of PNGase F; 3 and 5, periplasmic LecB (3) and HW LecB (5) with PNGase F treatment. (C) Time course of the deglycosylation of HW LecB. HW LecB was incubated with PNGase F for 16 h at 37°C. After incubation periods of 0, 3, 6, 9, and 16 h, samples that each contained 100 ng of HW LecB were taken off. Samples were analyzed by SDS-PAGE and Western blotting using a LecB-specific antiserum.