Figure 5
Expression and immunodetection of EhRAD51. A. Expression and purification of rEhRAD51-6x His-tagged protein. Bacterial proteins were separated through 10% SDS-PAGE and gels were stained with Coomassie blue. Lane 1, molecular weight markers; lane 2, non-induced bacterial extract (30 μg); lane 3, IPTG-induced bacterial extract (30 μg) before passing through the Ni2+-NTA affinity column; lane 4, affinity purified polypeptide from IPTG-induced bacteria extract. Arrowhead, 47-kDa rEhRAD51. B. Immunodetection of rEhRAD51 polypeptide. Western blot assays were performed using non-induced bacterial extract (lane 1) and purified rEhRAD51 (lanes 2 to 4). Lanes 1 and 2; anti-6x His tag antibodies; lane 3, preimmune serum; lane 4, specific rabbit antibodies raised against rEhRAD51. Arrowhead, 47-kDa rEhRAD51. C. Immunodetection of E. histolytica endogenous EhRAD51 by Western blot assays using specific anti-EhRAD51 antibodies. CE, cytoplasmic extracts; NE nuclear extracts. Lanes 1 and 2, non-irradiated (No UV-C) trophozoites; lanes 3 and 4, irradiated (UV-C) trophozoites (30 min after UV-C treatment). Upper panel: arrowhead, 41-kDa EhRAD51; asterisk, 46-kDa EhRAD51.Controls using anti-EhPAP and anti-actin antibodies (middle and bottom panels, respectively) are shown.