Analysis of surface proteins. Surface proteins were isolated from C. diphtheriae wild-type and mutant strains and subjected to SDS-PAGE (A), Western blotting (B), and 2-D PAGE (C). For SDS-PAGE 25 μg of protein prepared from strains ISS3319 (lane 2), Lilo1 (lane 3), ISS4060 (lane 4), and Lilo2 (lane 5) were applied per lane on a 10% polyacrylamide gel and silver-stained after electrophoresis. Molecular weight of marker proteins (lane 1, from top to bottom): 250, 130, 95, 72, 55, 36, 28, 17, 11 kDa. Western blotting was carried out after SDS-PAGE using a polyclonal antiserum directed against C. diphtheriae DSM44123 surface proteins. For 2-D PAGE surface protein preparations were separated according to their isoelectric point and molecular mass using a pH range of 3-10 for isoelectric focussing and 12.5% polyacrylamide gels for SDS-PAGE. Gels were stained with Coomassie Brilliant Blue. Molecular weight of marker proteins (from top to bottom): 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15 kDa.