*2.12. Immunodetection of (HA)-Tagged EhTrxR*

*E. histolytica* control and HA-tagged EhTrxR trophozoite cytosolic proteins (40 μg) were prepared according to a published method [36] and resolved on a 10% SDS-PAGE in SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). The resultant protein bands were visualized after staining with Ponceau-S (Sigma-Aldrich, USA). Next, proteins were electrotransferred in protein transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) to nitrocellulose membranes (Whatman, Protran BA83). The blots were first blocked using 3% skim milk and then probed with 1:500 mouse monoclonal HA antibody clone 12CA5 (a kind gift from Prof. Ami Aronheim) for 16 h at 4 ◦C. After incubation with the primary antibody, the blots were incubated with 1:5000 secondary antibody for one hour at RT (Jackson ImmunoResearch, PA, USA), and then developed using enhanced chemiluminescence (Bio RAD, Rishon Le Zion, Israel).
