FIGURE 2.

Co-purification of proteins that interact with YnjEΔ1–21 by purification using Protein G-Sepharose coupled with a YnjE antibody. A polyclonal antibody raised against YnjEΔ1–21 was bound to Protein G-Sepharose (GE Healthcare). A, His₆-YnjEΔ1–21 (40 μm) was incubated with 50 μm MoaD, 50 μm IscS, 50 μm MoeB, 50 μm MoaE, and 100 μm Mg-ATP (lane 1); Mg-ATP was omitted (lane 2); MoaE was omitted (lane 3); MoaD was omitted (lane 4); and IscS was omitted (lane 5). B, His₆-YnjEΔ1–21 (40 μm) was incubated with 30 μm MoeB (lane 1); 30 μm MoeB and 30 μm IscS (lane 2); and 30 μm IscS (lane 3). The mixtures in 100 mm Tris-HCl, pH 7.2, were applied to the beads, and after extensive washing steps, proteins were eluted with 500 mm citrate buffer, pH 2. Eluted proteins were concentrated and separated by 15% SDS-PAGE.