Immunoblot analysis of Mce4A protein. (A) Purified Mce4A protein separated by SDS-PAGE and electrotransferred to nitrocellulose membrane. Rabbit polyclonal antibody to Mce4A protein used at a dilution of 1:2000, detected signal of purified Mce4A protein. Lane 1-Pre-stained molecular mass marker (188 kDa, 62, 49, 38, 28, 18, 14, 6, 3, Invitrogen), the molecular mass standards are indicated in kDa on left side. Lane 2: Purified Mce4A protein (1 μg). Mce4A protein is indicated by arrow. (B) Subcellular fractions of M. tuberculosis H37Rv, stationary phase culture were fractionated on SDS-PAGE and Western blotting was performed using polyclonal anti-Mce4A antibodies for localization of Mce4A in various fractions. Concentration of protein loaded per well is indicated in parenthesis. Lane CL-Whole cell lysate (50 μg), Lane C-Cytoplasm (40 μg), Lane CFP-Culture filtrate proteins (30 μg), Lane CM-Cytoplasmic membrane (30 μg), Lane M-Pre-stained molecular mass marker (188 kDa, 62, 49, 38, 28, 18, 14, 6, 3, Invitrogen), the molecular mass standards are indicated in kDa on right side, Lane CW-Cell wall fraction (30 μg). Mce4A protein is detected only in the whole cell lysate and cell wall fraction from stationary phase culture.