B. burgdorferi BamA is present in OM protein complexes. A. The presence of BamA in OM complexes was revealed by blue native (BN)-PAGE analysis. OM proteins (20 μg) were separated by one-dimensional BN-PAGE (left panel). Subsequently, a strip of BN gel was excised and electrophoretically transferred, and immunoblot analysis was performed with anti-BamA antisera (right panel). Molecular weight standards, in kDa, are indicated at left. Arrows indicate two prominent bands resolving at ~200 kDa and 1000 kDa. B. Purity of a representative OM preparation used for BN analysis. B. burgdorferi protoplasmic cylinders (PCs) and OMs were isolated by sucrose density gradient centrifugation, as described in Methods. Cell equivalents of OM and PC fractions were separated by SDS-PAGE, electrophoretically transferred onto nitrocellulose membrane, and subsequently immunoblotted with antibodies against BamA and the periplasmic FlaB protein. As expected, BamA is present in the OM, while FlaB is enriched only in the PC fraction.