The column was eluted with water containing 0.1 % TFA (0 5 min), 40%% ethanol (5 10 min) followed by a linear gradient from 40 to 90%% ethanol in 5 min (flow rate 1 ml/min).
p11 was eluted by a linear gradient of (NH4 2SO4 from 50% to 0% and recovered in the last fractions.
The filtrate was injected onto a 2.1×250 mm C18 reverse-phase column (Vydac) and separated for 24 min. by a linear gradient of acetonitrile (23 35%) with 0.1% (v/v) trifluoroacetic acid as the ion-pairing agent.
A ten-minute rinse (at 5 ml/min) of the column with an aqueous solution containing 0.1%TFA was followed by a linear gradient (0 100%) elution over 150 min in a mobile phase containing 80% acetonitrile and 0.1%TFA.
During the first 7 min. the solvent composition was kept at 100% "A" followed by a linear gradient of 3 min. to 80% "B" and re-equilibration to the initial conditions.
The center of the green disk had a diameter subtending 1.5°, surrounded by a linear gradient between green and the background color that brought the overall blurred disk diameter to 2.25° visual angle.
A linear gradient from 0% to 15% Buffer B was run over 5 column volumes and it was followed by a linear gradient from 15% to 50% Buffer B over 25 column volumes to elute the TCTPs.
This consisted of the equiluminant cyan center, a dot of diameter 0.08°, surrounded by a linear gradient between cyan and black that brought the overall blurred dot diameter to 0.12° visual angle.
Bound antigen was eluted from the column by a linear gradient of 0 1 M NaCl and fractions were assayed for hexose content by the Dubois assay and for phosphorus using the Lowry method [36], [37].
After sample loading (10 mL, 6 mg/mL protein concentration), the column was washed with 10 volumes of buffer A (at 1 mL/min flow rate), followed by a linear gradient from 0.1 to 0.7 M NaCl over 210 minutes.
During the first 7 min the solvent composition was kept at 100% "A" followed by a linear gradient over 3 min to 80% "B" and re-equilibration to the initial conditions.
