2.5 Preparation of bacterial FAS for FAS inhibition assay
Crude FAS containing protein fraction was isolated according to the
modified protocol of Ahmad et al. (1981). Stock culture ofBacillus subtilis was inoculated in 100 mL LB broth pH 7.2 and
cultured for 24 hrs at room temperature. It was used for further
inoculation of four 1000 mL flasks with same media and culture
conditions. The flasks were placed in a rotator shaker to ensure
adequate suspension of cells. Bacteria were harvested during the
late-exponential phase of growth by centrifugation at 10000 x g for 10
min and washed twice with 50 mM sodium phosphate buffer (pH 7.0). Cell
pastes were stored at -20 °C until use.