Fig. 1. Detection of a cruzipain binding protein in T. cruzi epimastigotes lysates. Proteins from epimastigotes lysates, or recombinant human cystatin C used as a control, were diluted in SDS-sample buffer (without boiling) under non-reducing conditions. After running a 16% SDS-PAGE, the polypeptides were transferred to nitrocellulose membranes. Proteins that were capable of binding to cysteine proteases were identified by incubating the membranes with (A) cruzipain (10 µg/ml) in PBS, 2 mM EDTA, 1% BSA (w/v), 0.05% (v/v) Tween 20 for 1 hour at room temperature. After washing, the bound cruzipain probe was identified by treating the membrane with mAbcruzipain. Peroxidase-labeled anti-mouse IgG was used to visualize the immuno-reactive bands (B), membrane treated with buffer (instead of the cruzipain probe) followed by incubation with mAbcruzipain. Lane 1: recombinant human cystatin C (10 ng/ml); lane 2: epimastigote lysate.

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