Fractionation of F tularensis ssp. novicida whole-cell lysates into outer membrane, inner membrane, and cytoplasmic fractions was accomplished using the Sarkosyl membrane fractionation method as adapted by de Bruin et al. (2007) (link). Proteins were detected by separation on a 15% SDS polyacrylamide gel, followed by Western immunoblot using anti-FLAG M2 (Sigma) monoclonal antibody, rabbit polyclonal anti-Tul4 antisera, rabbit polyclonal anti-VgrG, or rabbit polyclonal anti-PdpB [gift from F. Nano; (Ludu et al., 2008 (link))], and ECL detection reagent (Amersham-Pharmacia).
For detection of lipid incorporation into IglE, strains were grown at 37 °C in CDM with [3H]palmitic acid (Moravek Biochemicals) to a final concentration of 20 μCi mL−1. Overnight cultures were pelleted, washed once with PBS, and then resuspended in 1X sample buffer and boiled. Samples were separated by 15% SDS-PAGE and then fixed with 5% glacial acetic acid, 5% isopropanol, and water. The gel was then treated with Autoflour (National Diagnostics) and imaged by autoradiography. Measurement of the labeled protein at 13.2 kD normalized to the label incorporated into a constant band at c. 16 kD in every lane was used to quantitate the relative labeled IglE levels by densitometry of the autoradiograph.
For detection of lipid incorporation into IglE, strains were grown at 37 °C in CDM with [3H]palmitic acid (Moravek Biochemicals) to a final concentration of 20 μCi mL−1. Overnight cultures were pelleted, washed once with PBS, and then resuspended in 1X sample buffer and boiled. Samples were separated by 15% SDS-PAGE and then fixed with 5% glacial acetic acid, 5% isopropanol, and water. The gel was then treated with Autoflour (National Diagnostics) and imaged by autoradiography. Measurement of the labeled protein at 13.2 kD normalized to the label incorporated into a constant band at c. 16 kD in every lane was used to quantitate the relative labeled IglE levels by densitometry of the autoradiograph.
