The TM123-MBP fusion protein is integrated into the membrane in the absence of the Tat machinery. Crude membranes were prepared from E. coli strains HS3018-A and HS3018-A ΔtatABC producing TM123-MBP, TM12_KK3-MBP, or _KKTM123-MBP as described in Materials and methods. The crude membranes were incubated with either 0.2 M Na₂CO₃ or 4 M urea followed by recovery of the membrane pellet by ultracentrifugation. The presence of the fusion protein in the wash supernatant (S) and pelleted membrane (P) was analyzed by immunoblotting using anti-MBP antiserum.