Borrelia burgdorferi Protein Co-IP

Co-IP experiments were performed using the Pierce Direct IP Kit (Thermo Scientific, Rockford, IL). Briefly, B. burgdorferi B31 cells were grown to mid-log phase (5 × 10⁷ organisms ml⁻¹) in complete BSK-II medium before whole-cell lysates were prepared as previously described (Lenhart et al., 2012 (link)). 500 µl of final lysates were then mixed with AminoLink resin with conjugated BB0794, BamA, or GST antibodies following the manufacturer’s instructions (Thermo Scientific). The lysate and resin mixture was rotated end over end at 4°C overnight before being washed. Proteins bound to the resin were eluted by adding 50 µl of final sample buffer containing β-mercaptoethanol and boiled for seven minutes before SDS-PAGE and immunoblot analysis.