Fig. 2. Detection of the cruzipain-chagasin molecular complex in vitro and in vivo. (A) The cruzipain-chagasin complex is stable upon treatment with SDS-ßME. Purified cruzipain and recombinant chagasin were incubated at equal concentrations in PBS, pH 7.2, for 20 minutes and subsequently diluted in different solutions: buffer alone (62.5 mM Tris-HCl pH 6.8, 10% glycerol) (lane 1); buffer containing 2% SDS; buffer containing 2% SDS and 5% ßME; and buffer containing 2% SDS and 5% ßME, and boiled for 5 minutes. The samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes and incubated with rabbit anti-chagasin antiserum (1:1000). (B) Free cruzipain is inactivated by SDS-ßME. The cruzipain-chagasin complex was formed as described in A and incubated in buffer containing 2% SDS and 5% ßME (lane 1) or cruzipain was diluted in the SDS-ßME buffer immediately before the addition of chagasin (lane 2). The samples were not boiled before loading in SDS-PAGE. Western blots with anti-chagasin antibodies were performed as described above. (C,D) Detection of the cruzipain-chagasin complex in living parasites. Epimastigotes (5x10⁶) were washed twice in PBS and lysed directly by addition of a solution containing 2% SDS and 5% ßME. The samples (not boiled) were resolved by SDS-PAGE and submitted to western blot with anti-chagasin antibodies (C) or anti-cruzipain anti-serum (1:1000) (D).