FIG. 5.

BldG and SCO3548 self-interactions were detected by far-Western analysis and affinity purification, respectively. (A) For far-Western analysis, cell lysates from S. coelicolor ΔbldG 1DB containing pIJ6902 (vector control) (lane 1) or pAU316 (bldG expression) (lane 2) were subjected to SDS-PAGE, and the separated proteins were transferred to a PVDF membrane. The membrane was incubated with purified GST-BldG, washed thoroughly, and probed with anti-GST antibody. (B) For affinity purification, cell lysate from E. coli BL21(DE3) containing pET30(a)⁺ (vector control) (lane 1) or containing pAU318 (His₁₀-SCO3548) (lane 2) was mixed with cell lysate from E. coli BL21(DE3) containing pAU376 (GST-SCO3548) at a ratio of 1:1 (vol/vol). Ni-NTA agarose was added to capture His₁₀-containing protein complexes, which were then eluted with 250 mM imidazole. Eluted proteins were analyzed by SDS-PAGE and Western analysis using anti-GST antibody. The positions of molecular mass markers are indicated on the left.