Fig. 1.

Fractionation of SGE. A, a diagram of the workflow for sample fractionation. B, SGE aliquot of 0.41 g was dissolved in 10 ml of 0.1 m phosphate-buffered solution, pH 6.0, and then was applied to a Sephadex G-75 (Superfine; Amersham Biosciences; 2.6 × 100 cm) gel filtration column equilibrated with 0.1 m PBS. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The absorbance of the eluate was monitored at 280 nm. C, SDS-PAGE analysis of factions eluted from Fig. 1B in 15% gel concentraion. 1–3: fractions 1–3 as indicated in Fig. 1B; R, reduced; UR, unreduced. D, fraction 2 from Fig. 1B was subjected to AKTA FPLC Mono Q (1 ml volume; Amersham Biosciences) anionic exchange equilibrated with 0.02 m Tris-HCl, pH 8.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. E–F, SDS-PAGE analysis of peaks 2.1 and 2.2 as indicated in Fig. 1C in 15% gel concentration, respectively. R, reduced; UR, unreduced. G, fraction 3 from Fig. 1B was subjected to AKTA FPLC Resource Q (10 ml volume; Amersham Biosciences) anionic exchange equilibrated with 0.02 m Tris-HCl, pH 8.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. H, peak 3.1 as indicated in Fig. 1G was subjected to AKTA FPLC Mono S (1-ml volume; Amersham Biosciences) cationic exchange equilibrated with 0.02 m PBS, pH 6.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. I, peak 3.2 as indicated Fig. 1G was purified further by RP-HPLC (Hypersil BDS C₄, 30 × 0.46 cm) column with the indicated acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid in water. J, peak 3.1.2 as indicated in Fig. 1H was purified further by RP-HPLC (Hypersil BDS C₄, 30 × 0.46 cm) column, and the elution was performed at a flow rate of 0.7 ml/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water. K and L, peaks 3.1.4, 3.1.5, 3.1.7–9 (tablysins 2–6) as indicated in Fig. 1H, and peaks 3.1.6 (Fig. 1H), 3.1.3 (Fig. 1H), 3.1.1(Fig. 1H), 3.2.1 (Fig. 1J), 3.2.3 (Fig. 1J) (tabinhibitins 3–7) were subjected to reduced SDS-PAGE analysis in a gel concentration of 15%, respectively. M, the peak 3.1.2.1 (tablysins 7) as indicated in Fig. 1J was subjected SDS-PAGE analysis in a gel concentration of 15%. R, reduced; UR, unreduced. N, fraction 5 as indicated Fig. 1B was purified by RP-HPLC (Hypersil BDS C₈, 30 × 0.46 cm) column, and the elution was performed at a flow rate of 0.7 ml/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water.