Figure 3
Purification of B6Fabenv12CS. Renatured material (2.3 litre) was subjected to a series of chromatographic purification columns as described in methods. The fractions obtained through each purification step were analysed by electrophoresis on 0.1% SDS-10% PAG under non-reducing conditions. Each lane contains sample equivalent to 10 μl of each fraction. The protein bands were visualised with Coomassie blue R-250 staining. The figure shows Coomassie blue stained gel of the eluted fractions from (A) SP-Sepharose, (B) Q-Sepharose, and (C) Sephacryl S-200 columns. Panel (A): Lane D, dialysed renatured material; Lane 23–37, fractions 23 to 37 of SP-Sepharose column; Lane C, Purified B6Fab protein (control); Panel (B): Lane P1, pool of fractions from SP-Sepharose column; Lane 22–38, fractions 22 to 38 of Q-Sepharose column; Panel (C): Lane P2, pool of fractions from Q-Sepharose column; Lane 65–79, fractions 65 to 79 of Sephacryl S-200 gel filtration column. Filled arrows indicate the position of B6Fabenv12CS protein and hollow arrows of free B6LC. Agglutination activity of the fraction before addition of 9E10 (Ab) and after addition of 9E10 (Aa) are shown in boxes. '0' indicates no visible agglutination. The number indicates maximum dilution of sample that gave visible agglutination.