Gag-binding activity of artificial ankyrins. (A), Competition ELISA. Samples of biotinylated Gag-binders AnkGAG1B8, AnkGAG1D4 and AnkGAG6B4, and of control biotinylated αRep-A3-binder AnkA32D3 were mixed with their corresponding non-biotinylated form (black bars), or mixed with irrelevant soluble target (grey bars), or mixed with buffer containing no inhibitor (white bars). Mixtures were added to H6MA-CA- or αRep-A3-coated wells, as indicated at the bottom of the panel. Bound-ankyrins were detected by addition of HRP-conjugated extravidin, followed by the TMB substrate. (B), Far Western blotting. Lysates of BV-H6MA-CA-infected Sf9 cells were electrophoresed in SDS-gel, proteins transferred to a PVDF membrane, and membrane cut into strips. Gag-binding activity was determined by incubation of the strips with the different biotinylated ankyrins AnkGAG1B8, AnkGAG1D4, AnkGAG6B4, and AnkA32D3, as indicated on top of the strips. On the rightmost strip, the respective positions of the Gag proteins H6MA-CA and H6MA were determined using anti-histidine tag antibody (arrowheads). (C), Indirect ELISA. H6CA was captured on nickel-coated plate, and used as substrate for binding assay of biotinylated ankyrins AnkGAG1B8, AnkGAG1D4, AnkGAG6B4, and AnkA32D3. Bound-ankyrins were quantitated as in (A). BG, background signal.