FIG. 3.

Purification of Giardia eIF4E2 by m⁷GTP-Sepharose affinity chromatography and identification by Western blotting. (A) Cell lysate from 2 liters of Giardia cell culture was applied to an m⁷GTP-Sepharose column and eluted. Proteins in collected fractions (lanes: 1, total protein; 2, flowthrough; 3, column wash; 4, column washed with GTP; 5, column eluted with m⁷GTP) were separated by SDS-PAGE and visualized by E-zinc staining. (B) The eluted fraction from an m⁷GTP-Sepharose affinity column separated by SDS-PAGE (Fig. 2A, lane 5) was transferred onto a polyvinylidene difluoride membrane and subjected to immunoblotting with purified rabbit polyclonal antibodies against Giardia eIF4E1 and eIF4E2, respectively. Purified recombinant Giardia eIF4Es (see Fig. S1 in the supplemental material) were included in various quantities as a control.