Mycobacterial cell fractionation.

Mycobacteria cell fractionation was done as described elsewhere (52). Briefly, the cells were lysed in a buffer that contained 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 20 mM KCl, 10 mM MgCl₂. Bacterial culture was homogenized with a Minilys homogenizer (Bertin Instruments) using glass beads. A cocktail of proteinase/phosphatase inhibitors (Roche, UK) were used in all buffers. Lysates were centrifuged for 1 h at 27,000 × g, and the pellets were washed in a carbonate buffer (pH 11) and used as cell wall material. The supernatant was centrifuged again for 4 h at 100,000 × g. The supernatant from this step was used as cytoplasmic fraction, and the pellets (membrane fractions) were washed once in carbonate buffer, pH 11, and twice in Tris-buffered saline (TBS) buffer. Proteins from cellular fractions were separated on SDS-PAGE. The purity of fractions was confirmed by the detection of diagnostic proteins as described below.