Figure 4

Obg cofractionation with ribosomal subunits. M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C, and lysates prepared using a bead beater. About 500 g protein was separated in 10-40% sucrose gradient. A. The ODs of the separated fractions were measured (manually) at 260 nm. B. The proteins in the fractions were then precipitated with ethanol and separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Obg antiserum (1:500 dilution), followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. Lane C is a whole-cell extract from M. tuberculosis. Lanes 1-15 represent fractions from the top (10% sucrose) to the bottom (40% sucrose) of the sucrose gradient. Fraction 16 was not analyzed in immunoblot.