Western blot analysis. Flagellin of SL5928(fliC) and chimeric flagellin of SL5928(fliC/esat) were extracted by acid treatment followed by ammonium sulfate precipitation. (a) The crude extract of fliC protein and chimeric flagellin fliC/esat were subjected to SDS–PAGE (M, standard molecular weight markers in kDa). Lane 1, crude extract of flagellin of SL5928(fliC); lane 2, crude extract of chimeric flagellin of SL5928(fliC/esat). (b) The crude extract of fliC protein and chimeric flagellin fliC/esat were transferred onto nitrocellulose and treated with anti-ESAT-6 antibodies (mAb HYB 076–08). A prominent 52 kDa band was observed (lane 4). Lane 3, crude extract of flagellin of SL5928(fliC); lane 4, crude extract of chimeric flagellin of SL5928(fliC/esat). mAb, monoclonal antibody; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.