FIG. 1.

Electromobility of shed GXM from JEC21 cells changes over time in rich medium. All samples were heated (15 min at 70°C) to denature enzymes, centrifuged (16,000 × g for 3 min) to separate supernatant and cells, and stored at 4°C. Samples (15 μl) of culture supernatants were mixed with 6× DNA loading dye (4), loaded on a 0.6% certified megabase agarose (Bio-Rad) gel, and subjected to electrophoresis (15 h at 25 V) in 0.5× TBE (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA, pH 8.3). Samples containing more GXM (later time points) were diluted in distilled water based on a pilot gel, as sample normalization based on enzyme-linked immunosorbent assay determination of GXM concentration (23) did not yield equal blot intensity. (This may reflect changes over time in GXM antibody reactivity [7, 11, 18] and/or transfer efficiency.) The gel was transferred onto a positively charged nylon membrane and immunoblotted with 1 μg/ml anti-GXM antibody 3C2 as described in the text. Initial sample heating and dilution in distilled water did not alter electrophoretic migration (data not shown). St, starter culture (at 1.6 × 10⁷ cells/ml); arrowhead, well position; arrow, direction of migration; other numbers, sampling time (hours) after the start of the experiment.