FIG. 1.
ImpLM is an integral inner membrane protein. (A) Equal volumes of total proteins (TP), periplasmic proteins (P), cytoplasmic and membrane proteins (CM), cytoplasmic proteins (C), and membrane proteins (M) of wild-type strain A. tumefaciens C58 grown in AB-MES (pH 5.5) for 6 h at 25°C were resolved by 12% glycine-SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were analyzed by immunoblotting with antibodies against C-ImpLM and ActC, which served as a soluble protein marker. (B) Membrane fractions separated by sucrose density gradient centrifugation were collected from the top of the gradient and analyzed by immunoblotting with C-ImpLM antibody. The fractions containing the outer membranes (OM) and inner membranes (IM) were identified on the basis of the activity of the inner membrane marker NADH oxidase. The values for sucrose density and A280 for each of the fractions of the 53% to 70% sucrose gradient are indicated. (C) Total membranes were incubated with various chemical reagents and centrifuged to separate soluble (S) and pellet (P) (insoluble) fractions. The fractions were analyzed by immunoblotting with C-ImpLM antibody. The positions of the molecular mass markers used (in kDa) are indicated on the left in each panel. TX-100, Triton X-100; LS, N-lauroylsarcosine.